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Racchetti et al. Extracell Vesicles Circ Nucleic Acids 2023;4:44-58 https://dx.doi.org/10.20517/evcna.2023.03 Page 48

remodeling by interacting with vacuolar protein sorting (VPS) and an ATPase [22-25] . Fission occurs when
ESCRT-III is removed, leading to complete separation of the exocytic neck and ensuing exosome formation
within the MVB lumen [Figure 1].

Subsection 2C: Exosome cargoes [Figure 1]. The accumulations of cytoplasm within small initial MVB
protrusions begin cargo growth. Some cytosolic proteins are typical of these cargoes. Its surface proteins are
often anchored (by myristoylation, palmitoylation, or other sequences) to the luminal membrane surface of
exosomes. Other proteins, including the ESCRT-associated TSG 101, ALIX, and some HSPs, participate in
post-translational modifications and complex assembly [21,27] , contributing to cargo growth [27-29] . Proteins are
not the only components of cargo, which contain molecules of different natures. There are also small
sequences of DNA, lipids, and metabolic molecules; highly abundant are various types of RNA (mostly
microRNAs, miRs, together with messenger RNAs, long non-coding RNAs, and ribosome RNAs). The
cargo molecule presence is relevant. In fact, RNA-binding proteins undergo condensation [25,27] . Together
with essential factors such as IL-1β and TNF-α, various proteins undergo selective engulfment within
exosomes [30,31] . In addition, recent results have confirmed the relevance of protein-RNA binding. Condensed
YBX1 proteins induce liquid-liquid phase separations in cargoes, recruiting miR-223 and enabling their
targeting and packaging within growing exosomes . Further details have been clarified by the
[31]
identification, active in the assembly of cargoes, of proteins containing endofin (i.e., a protein domain
confined to endosomes, and of ARRDC1, an adapter of ubiquitin ligases) [30,32] . Moreover, vesicle cargoes
[33]
accumulate additional factors by a transport process dependent on another protein, LAMP2A . Finally, the
interest in cargoes should also be focused on unconventional secretion processes (UPS). Many involved
proteins are loaded into exosomes [34-36] .

Subsection 2D: Journey of MVBs and exosomes. Once heavily loaded by numerous exosomes, MVBs travel
within the cell. In response to various types of stimulation they move, approaching the plasma membrane.
[37]
Upon tethering to specific sites , there is some heterogeneity of the molecules participating in MVB
exocytosis. The first Ras GTPase active in the process is Ras11. Additional GTPase forms, such as Rab27a
and Rab 27b and members of the Rho, Rac, and cdc42 family, have been reported to operate in many, but
not all, cell types [38,39] .

Concerning exocytic fusion, the protein most frequently involved in various tissues and cancers is R-
SNARE VAMP7 with Q-SNARE SNAP23 [40,41] . However, other R-SNAREs (including VAMP3 and VAMP8)
are also effective with lower frequency [42,43] . Their ternary complex, established with SNAP23 associated with
syntaxin-4, induces the generation of enlarging pores, called invadopodia, critical sites for MVB fusion with
the plasma membrane and the subsequent exosome release [40-43] . Small GTPases include Ral, Rab (especially
Rab35), and other Ras . The integrated analysis of the various participants has revealed the role of non-
[44]
coding RNAs and G protein-coupled receptors [43,44] . The latter, via their cAMP effect, promote the fusion via
[43]
a SNAP23 phosphorylation at the Ser 110 position .
Recent developments in pH-dependent fluorescence microscopy revealed exocytoses’ frequency,
localization, and machinery. Exosome localization experiments have revealed unexpected results. In
[35]
lymphocytes, the site of exocytosis is redistributed upon the establishment of immune synapses . In
epithelial cells, MVB exocytoses addressed to the basolateral area differ from those addressed to the apical
area. Differences have also been demonstrated between the two corresponding families of released
exosomes [39,45] . Therefore, heterogeneity is a common property of exosomes discharged even by single cells.
Finally, recent evidence has demonstrated that the release and distribution of exosomes in the brain plays an
unexpected, critical role in the pathogenesis of neurodegenerative diseases, a new role that will be