Exosomes are a type of extracellular vesicle released from cells after fusion of multivesicular bodies with the plasma membrane. These vesicles are often enriched in cholesterol, SM, glycosphingolipids, and phosphatidylserine. Lipids not only have a structural role in exosomal membranes but also are essential players in exosome formation and release to the extracellular environment. Our knowledge about the importance of lipids in exosome biology is increasing due to recent technological developments in lipidomics and a stronger focus on the biological functions of these molecules. Here, we review the available information about the lipid composition of exosomes. Special attention is given to ether lipids, a relatively unexplored type of lipids involved in membrane trafficking and abundant in some exosomes. Moreover, we discuss how the lipid composition of exosome preparations may provide useful information about their purity. Finally, we discuss the role of phosphoinositides, membrane phospholipids that help to regulate membrane dynamics, in exosome release and how this process may be linked to secretory autophagy. Knowledge about exosome lipid composition is important to understand the biology of these vesicles and to investigate possible medical applications.

Exosomes are small extracellular vesicles released to the extracellular milieu through fusion of multivesicular bodies with the plasma membrane. These vesicles contain microRNAs and might therefore be vehicles transferring genetic information between cells. The aim of this study was to investigate whether there was a sorting of microRNAs into exosomes in the prostate cancer cell line PC-3. In addition, microRNAs in PC-3 cells and in the non-cancerous prostate cell line RWPE-1 were compared. Exosomes were isolated from the conditioned media from PC-3 cells by ultracentrifugation and inspected by electron microscopy. Total RNA was isolated and microRNAs were analyzed by microarray analysis and real time RT-PCR. MicroRNA microarray analysis revealed that the microRNA profile of PC-3 released exosomes was similar to the profile of the corresponding parent cells. Nevertheless, a sorting of certain microRNAs into exosomes was observed, and low number microRNAs (microRNAs with a low number in their name) were found to be underrepresented in these vesicles. Moreover, the miRNA profile of PC-3 cells resembled the miRNA profile of RWPE-1 cells, though some miRNAs were found to be differently expressed in these cell lines. These results show that exosomes from PC-3 cells, in agreement with previous reports from other cell types, contain microRNAs. Furthermore, this study supports the idea that there is a sorting of microRNAs into exosomes and adds a new perspective by pointing at the underrepresentation of low number miRNAs in PC-3 released exosomes.

Cells release extracellular vesicles (EVs) of different sizes. Small EVs (< 200 nm) can originate from the fusion of multivesicular bodies with the plasma membrane, i.e. exosomes, and from budding of the plasma membrane, i.e. small ectosomes. To investigate the molecular machinery required for the release of small EVs, we developed a sensitive assay based on incorporation of radioactive cholesterol in EV membranes and used it in a siRNA screening. The screening showed that depletion of several SNARE proteins affected the release of small EVs. We focused on SNAP29, VAMP8, syntaxin 2, syntaxin 3 and syntaxin 18, the depletion of which reduced the release of small EVs. Importantly, this result was verified using gold standard techniques. SNAP29 depletion resulted in the largest effect and was further investigated. Immunoblotting analysis of small EVs showed that the release of several proteins considered to be associated with exosomes like syntenin, CD63 and Tsg101 was reduced, while the level of several proteins that have been shown to be released in ectosomes (annexins) or by secretory autophagy (LC3B and p62) was not affected by SNAP29 depletion. Moreover, these proteins appeared in different fractions when the EV samples were further separated by a density gradient. These results suggest that SNAP29 depletion mainly affects the secretion of exosomes. To investigate how SNAP29 affects exosome release, we used microscopy to study the distribution of MBVs using CD63 labelling and CD63-pHluorin to detect fusion events of MVBs with the plasma membrane. SNAP29 depletion caused a redistribution of CD63-labelled compartments but did not change the number of fusion events. Further experiments are therefore needed to fully understand the function of SNAP29. To conclude, we have developed a novel screening assay that has allowed us to identify several SNAREs involved in the release of small EVs.