Biogenesis of exosomes occurs by inward budding of the plasma membrane, which forms the endosome vesicle and multivesicular bodies (MVBs). MVBs fuse with lysosomes, degrade or fuse with the plasma membrane and form exosomes that are released from the cells into the extracellular space (Gaurav I et al. 2021). Late endosomal structures containing numerous intraluminal vesicles (ILVs) are known as multivesicular bodies, which are eventually transported to the trans-Golgi network for endosome recycling, delivered to lysosomes for degradation of all entrained material, or fused to the plasma membrane and release exosomes into the extracellular space; this process is facilitated by Rab GTPases such as RAB11 and RAB35, which release exosomes enriched with flotillin and other cell-specific proteins (Yue B et al. (2020).
Exosome biogenesis and secretion thus requires the formation of an endosomal sorting complex required for transport (ESCRT) (Patil AA et al. 2019). The ESCRT consists of four complexes (ESCRT-0, ESCRT-I, ESCRT-II and ESCRT-III) and associated proteins (VPS4, Tsg101 and ALIX). ESCRT-0 sorts ubiquitinated cargo proteins into the lipid domain; ESCRT-I and ESCRT-II cause membrane deformation to form the stable membrane neck, and recruitment of the Vps4 complex to ESCRT-III leads to cleavage of the vesicle neck and dissociation and recycling of the ESCRT-III complex (Patil AA et al. 2019).
In contrast to ESCRT-sorted proteins, loading of RNA into exosomes is mediated by lipids and depends on self-assembling lipid and carrier motifs. Specific nucleotide sequences exhibit enhanced affinity for phospholipid bilayers depending on variables such as lipid morphology, hydrophobic changes and physiologically concentrated sphingosine in rafting membranes (O'Brien K et al. (2020).
Interestingly, the lack of ESCRT machinery did not prevent the formation of MVB vesicles in mammalian cells, but leads to reduced processing of their cargo and changes in number and size (Hessvik NP et al. 2018).
The methods of penetration of exosomes into recipient cells have not yet been sufficiently researched. However, it has been shown that, depending on the type of recipient cell, exosomes enter the target cells via fusion with the plasma membrane, macropinocytosis, phagocytosis and clathrin-dependent endocytosis.