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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 46

structures [11,12] . Our knowledge of the four membrane-bound structures shows this to be their case. MVBs
and their exosomes are largely endocytic. They originate from endocytic cisternae. Ectosomes are generated
[5,6]
from plasma membrane microdomains, including endocytic steps, as shown by specific markers .
Lysosomes competent for exocytosis, officially called endo-lysosomes, are generated by their fusion with
early-generated endocytic structures . Competent autophagosome fractions are activated by prefusion
[7,8]
with endocytic vesicles during maturation [9,10] .

In the present review, we intend to illustrate the role of the four intracellular membrane-bound structures,
from their generation to the discharge of their vesicles, including all steps before extracellular navigation.
The properties and functions of one type of these structures will be illustrated in a separate section, from
numbers two to five. In contrast, endocytic structures are not presented in a single section. The endosomes
are included in the corresponding sections.

MVBS AND THEIR EXOSOMES
In the introduction, we presented MVB, an endocytic cisterna that, by the inward budding and pinching off
of its small membrane domains, induces the generation and luminal accumulation of small vesicles, the
exosomes. This section is organized into four subsections. The first (2A) is focused on the properties and
functions of the whole MVB and has substantial relevance for cell function. Two subsections (2B and 2C)
illustrate the generation and properties of specific exocytic vesicle membranes and luminal cargoes. The
processes of MVB occurring upon the accumulation of their vesicles (i.e., their intracellular traffic leading
some toward the plasma membrane) and their exocytoses, are reported in subsection 2D. The role of MVB
and exosomes in diseases, especially neurodegenerative and cancers, have been illustrated in several reviews,
including ours [13,14] . Therefore diseases are not presented in this review.

Subsection 2A: MVBs as a whole. Exosome loading within MVBs depends on sirtuin2, a deacetylase
enzyme that participates in several other processes, including protecting neurons from neurodegeneration
[15]
and stimulating the viability of cancer cells . During and upon their vesiculation, MVBs undergo
maturation. They move within cells with substantial accumulation in the microtubule organization center.
From there, they move alternatively in two directions. Upon interaction with the Rab7 ortholog Ypt2 and
the multi-subunit tethering complex HOPS, some MVBs proceed to specific fusion with lysosomes
governed by the Qa-SNARE Pep12 protein. Upon such fusion (which accounts for a significant fraction of
the MVBs present in the cell), exosomes are discharged into the lysosome lumen, then disassembled, and its
[16]
components are exposed to catabolism. These exosomes will never be converted into EVs .
The remaining MVBs undergo exocytosis [Figure 1]. When lysosomes are disrupted, the alternative
exocytoses are significantly increased [17-19] . The initial step of the process is the MVB movement toward the
cell surface, increased by stimulatory signals. MVBs include a form of cooperation established with another
structure of vesicle generation, the autophagosomes. Comparative studies with and without autophagy
inhibitors have revealed the importance of this process. When autophagosomes are unavailable, exocytoses
of MVBs are significantly reduced; when autophagosomes are abundant, exocytoses are increased . The
[20]
integration of these two structures (illustrated in detail in subsections 5B and 5D on autophagosomes)
regulates some form of MVB exocytosis.

Subsection 2B: Exosome membranes [Figure 1]. The exosome membranes contain phospholipids and
lysobisphosphatidic acid (LBPA), an atypical phospholipid absent in many other types of membranes,
cholesterol and ceramide . The tetraspanins are abundant (in various forms, predominantly CD63) and
[21]
critically crucial for exosome assembly. Also essential are the integrins that convert signals across the vesicle