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Page 247 Raposo et al. Extracell Vesicles Circ Nucleic Acids 2023;4:240-54 https://dx.doi.org/10.20517/evcna.2023.18

the mechanistic basis of EV biogenesis opens the door to understanding EV secretion and the targeting of
signaling molecules to recipient cells. Cargoes that are selected by the aforementioned molecular
[68]
machineries can be post-translationally modified for selective recognition . Ubiquitination does not
appear to be required for sorting of MHC class II molecules to secretory MVEs, although ubiquitinated
proteins are commonly found in EVs . Interestingly, ESCRT components that sort proteins in MVEs
[69]
[54]
contain Ubiquitin Recognizing Motifs . Not ruling out ubiquitin completely, there are many forms of
ubiquitin conjugation including mono-ubiquitination involved in targeting CD133 to EVs . Other post-
[70]
translational modifications, such as palmitoylation and farnesylation, which bring proteins to lipid rafts, are
also implicated in EV biogenesis . The selective packaging of RNA into nascent EVs has seen significant
[68]
progress with the discovery that some miRNAs contain a targeting sequence recognized by a sumoylated
hnRNPA2B1 and that YBX-1proteins may selectively recruit specific miRNAs into newly forming
[71]
[72]
exosomes. Given the wide collection of RNA molecules that may be packaged as cargoes, different sorting
machineries may be exploited possibly by post-translational modification (e.g., ubiquitination,
palmitoylation, etc.) . Based on the potentially wide variety of sorting mechanisms and their role in
[73]
biogenesis, several questions arise: do all sorting mechanisms act on the same compartments, do they act
sequentially at different stages in maturation of these compartments, or do they act on distinct
compartments with unique features and fates. Of note, in endosomes, the cargoes and the sorting
machineries could also recruit or exclude additional machineries that regulate lysosomal fusion, MVE
transport to the plasma membrane, or fusion . One should also keep in mind that the budding step by itself
[7]
will change the composition of the limiting membrane and could lead to the recruitment of these regulatory
components. Lastly, EVs are secreted by polarized epithelia from both the apical side as well as the
basolateral domain . Recent work suggests that different EV sorting and secretion mechanisms may be at
[74]
play at these two locations . Apical secretion and basolateral secretion of EVs are likely to have widely
[75]
disparate functions.

CELL-CELL CONTACTS AND EVS WITHIN TISSUES, IMPACT OF THE
MICROENVIRONMENT
Close encounters within the tight quarters of the tissue environment raise many questions about how cells
communicate with each other: humoral (paracrine, autocrine), tubule (nanotubes), or vesicular [76,77] . While
the budding from the plasma membrane seems to be a relatively simple event and exosome secretion a more
regulated process, tracking the role of EVs in a 3D microenvironment raises key questions, including cell
type heterogeneity and the effect of external factors [7,78] . Within tissues, cells in contact with each other can
communicate via receptors and by direct contacts that can be maintained by filopodia and nanotubes . In
[77]
light of this, the need for a cell-free membrane-bound vesicle may be questionable. However, the very
selective packaging of cargo within EVs, independently of their origin, is likely to allow for communication
at short distances between cells while allowing for longer-range signaling within a tissue . Additionally,
[61]
physical parameters such as external pH, osmotic pressure, and physical constraints due to the matrix
structure of 3D environment including mechanical stress may affect EV secretion and targeting .
[79]

Several examples may serve as a kind of intellectual hors d’oeuvre to capture exciting developments in EVs
operating in the microenvironment. Contacts between cells, such as that observed during immune synapse
formation, elicit an increase in EV secretion and remodeling of the endocytic organelle to generate a
subpopulation of secretory MVEs . An interesting recent report highlights the potential importance of EVs
[65]
in the immunological synapse . Lanna et al. showed that antigen-presenting cells (APCs) extend the
[80]
lifespan of T cells that they form synapses with by transferring telomeric DNA from the APC to the T cell
via EVs. Telomeric DNA in APCs is trimmed away from its chromosomal localization and packaged in EVs
that are then secreted and delivered to the recipient T cells. These findings may be a harbinger of a new