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Page 49 Racchetti et al. Extracell Vesicles Circ Nucleic Acids 2023;4:44-58 https://dx.doi.org/10.20517/evcna.2023.03

[46]
investigated in the near future .

ECTOSOMES WITH THEIR SHORT INTRACELLULAR LIFE
Knowledge of ectosomes (more limited than exosomes) has been questioned for many years. Ectosome
fractions, recovered from EV mixtures, have been found in lower numbers (sometimes much lower) than
exosomes [5,6,31,46] . However, in a few studies, the fractions of the two vesicles are close [47,48] . The prevalence of
exosomes is, therefore, frequent; however, it is not the rule. Therefore, the comparison of the two vesicles
should be considered (and possibly established by advanced techniques) in EV mixtures investigated in
various conditions.

During the past decade, information about ectosomes has increased significantly. The mechanisms of their
generation differ substantially from those of the other vesicles, e.g., there are no exocytoses such as those of
the other membrane-bound structures. Instead, there are outward budding and vesicle release from the
external surface of the plasma membrane to the extracellular space [Figure 1]. Interestingly, the
microdomains involved in ectosome generation are not always spread in flat plasma membrane areas. Some
are concentrated over cell protrusions, such as filopodia and microvilli, operating as specialized platforms
[49]
for vesicle budding . Even if these processes are particular, some pre-EV properties (subsection 3A), i.e.,
the generation of the vesicle membranes (subsection 3B) and the assembly of their cargoes (subsection 3C),
are somewhat analogous to those of exosomes presented in section 2. Also analogous is our decision to omit
the role of ectosomes in diseases from this review. This decision has already been discussed in section 2
regarding exosomes [13,14] .

Subsection 3A: Intracellular life. The life of ectosomes is much shorter than that of other vesicles.
Intracellular ectosomes exist only during their generation, growth, and release; then, they are considered
among the EVs. Their plasma membrane microdomains, i.e., the sites of ectosome generation, appear
different from the rest of the plasma membrane. For example, the asymmetric phospholipid layers of their
membrane are rapidly rearranged. Several membrane proteins are analogous (however not identical) to
those of exosomes. For example, the most abundant tetraspanin of ectosomes is CD9 , not CD63, which
[48]
predominates in exosomes [21,48] . Proteins typical of the plasma membrane are present in ectosome
membranes during and after their generation; however only at low concentrations . In cells stimulated by
[50]
appropriate receptor agents (such as ATP), ectosome generation/release starts within the first few
minutes [50,51] . Cdc42 is a small G protein of the Rho family and a convergent node of multiple regulatory
signals. The binding to its downstream effector Ras GTPase-activating-like protein 1 is required for
ectosome shedding [52,53] . Additional stimulatory events sustained by LINK1 (a kinase that controls the
dynamics of actin cytoskeleton) are upregulated by RhoA and Rock. Inhibitors of these G proteins suppress
the production of ectosomes [53,54] . Subsequent developments have been confirmed by the involvement of
ESCRTs and their associated proteins [54,55] analogous (but not identical) to those of exosomes reported in
Subsection 2A.

Subsection 3B: ectosome membranes [Figure 1]. Two primary membrane processes activate typical
ectosome generations. Upon establishing plasma membrane microdomains, the first involves their
dynamics, with ensuing outward budding and fission [54,55] [Figure 1]; the second is based on the ESCRT-III
complex, followed by the appropriate ATPase, governing the increasing curvature of growing vesicles. The
ensuing narrowing of their neck, followed by their final scission, is followed rapidly by the release that
converts ectosomes into released EVs . These and other processes of membrane regulation include various
[48]
processes such as protein phosphorylation and calmodulin activation . Based on its properties, the
[56]
intracellular life of ectosomes appears to play a critical role in the regulation of vesicle biology .
[57]