**3. Lipid rafts and exosome biogenesis**

There are a number of lipid raft proteins that are associated with exosome biogenesis. Ceramide is enriched in the inner leaflet of the exosomal membrane [12]. One feature of ceramides is that they can initiate spontaneous membrane invagination which allows for ILV formation and the maintenance of vesicle shape and structure. One study showed exosome release was reduced after the inhibition of the enzyme that catalyzes the formation of ceramide from sphingomyelin, neutral sphingomyelinases [13]. Cholesterol is another lipid raft protein that is enriched in the exosomal membranes. The accumulation of cholesterol leads to the secretion of exosomes enriched in flotillins. In some cells the inhibition of sphingomyelinase results in the suppression of exosome production while exosome production is not affected by changes in sphingomyelinase activity in other cells. This suggest that there may be cell type specific mechanisms that regulate exosome production. For example, the ESCRT dependent mechanism may be favored over the lipid raft dependent mechanism in PC-3 cells since exosome production is not affected by inhibition of sphingomyelinase in these cells.

There are several other proteins that are involved in exosome biogenesis. Proteins associated with the ESCRT dependent pathway, glycosylphosphatidylinositol anchored proteins (GPIAPs), palmitoylated forms of transmembrane proteins, flotillins, annexins, and cholesterol binding proteins such as caveolins have been shown to be enriched in exosomes. Flotillin 1 and Flotillin 2 appear to play a role in EV composition as Phuyal et al. showed interfering RNA-mediated knockdown of flotillin 1 and flotillin 2 resulted in alteration of the EV composition in PC-3 cells [14]. The exosomal protein Annexin A2 may play an essential role in the organization of the exosomal membrane as it preferentially binds to phosphatidylinositiol 4,5 bisphosphate rich domains and cholesterols within the cytoplasmic leaflet of lipid raft membranes and influence raft dynamics of parent cells [15]. It is not surprising caveolins were found to play an essential role in EV generation and uptake since they are known to regulate multiple cellular processes including endocytosis, exocytosis,

and maintaining the shape of the cell membrane. Accumulating data suggest caveolin 1 promotes the production and release of EVs while caveolin deletion results in a decrease in EV release [16].
