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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 50

Subsection 3C: Ectosome cargoes [Figure 1]. Knowledge about ectosome cargoes is limited. Their
accumulation of proteins with high affinity binding to micro RNAs is similar to exosomes [28,31] (subsection
2C). Among such proteins is ARRDC1, a ubiquitin ligase adapter that regulates ectosome generation and
[58]
release . Loading of ectosome cargoes by RNA-binding proteins and miRNAs is supported by the LC3-
conjugated machinery, an example of vesicle/autophagy interaction . The ectosome cargo formation
[59]
requires some regulation by caveolin-1, a structural protein typical of plasma membrane caveolae, which is
also abundant in ectosomes; in contrast, caveolin-1 is not abundant in exosome cargoes . Additional
[56]
components of ectosome cargoes are IL-1β and other cytokines and factors of the TNF family with proteins
destined to be secreted by UPS . Upon their release from the plasma membrane, the ectosomes, not yet
[60]
fully distinguished from the other EVs, remain associated with the cell's surface (however, for short times
only).

NEWLY-DISCOVERED LYSOSOME FUNCTIONS
As already anticipated in the introduction, for decades the only function recognized for lysosomes was
passive digestion induced by their uptake of large numbers of molecules and organelles. In recent years,
additional specialized functions have been discovered and characterized, governed by their cooperation with
members of the endocytic system mediated by the Rab11 GTPases . These results have demonstrated the
[61]
[7]
multiplicity and complexity of lysosomal functions . These properties will be presented in three
subsections: 4A, covering endo-lysosomes; 4B, dealing with cargoes; and 4C, covering lysosomal disorders.

Subsection 4A: The endo-lysosomes. The new identification of lysosomal functions emerged unexpectedly
upon the discovery of endo-lysosomes, a form of organelle fused with an early endocytic structure of
considerable surface extension . The investigation of this innovative form of lysosomes revealed the co-
[62]
expression of properties typical of endosomes, including various forms of flotillin, several cargo proteins,
the TPC2 cation channels, and others [63-66] . Several functions have been reported to depend on endo-
lysosomes. The first were nutrient sensing, intracellular signaling, and intracellular metabolism [61,62] . More
recently, endo-lysosomes revealed additional processes such as intracellular trafficking, lysosome pH
regulation, fusion/fission with other organelles, and examples of cell secretion [63-66] . For the present review,
the endo-lysosome function of highest interest is exocytosis established by fusion with the plasma
membrane [Figure 2]. Markers of this process can be the appearance at the cell surface of luminal lysosomal
epitopes such as LAMP1 [67-69] , followed by an endocytic recycling response, activated to compensate the
excess of surface space/composition increase induced by the endo-lysosome exocytosis [68,69] . The latter is a
2+
Ca -dependent process controlled by the G proteins Rab11a and Rab11b, oriented toward the plasma
membrane. Rab11s interact with other molecules, such as the guanine nucleotide exchange factor GRAB
and Rab3a [61,67] . Thus, endo-lysosome exocytosis is a precisely controlled process.

Subsection 4B: Endo-lysosome cargoes [Figure 2]. A critical aspect of endo-lysosome exocytosis is the
nature of cargo components discharged extracellularly [63,68] . Within lysosomes, the structures are at least
partially digested. However, some conserved complexes, such as tetrameric and heterodimeric structures,
resist digestion . Enzyme release (in particular of a proteomic nature) can affect the structure of the
[63]
extracellular matrices. In addition, endo-lysosome exocytoses have been reported to release vesicles,
including some accumulated upon previous fusions with MVBs and autophagy [Figure 2]. Therefore, there
is a chance for endo-lysosome exocytosis releasing a small fraction of operative EVs [63,68] .

Subsection 4C: Effects on other organelles induced by lysosome disorders. A final question about
lysosomes deals with its lysosome storage disorders (LSDs) induced by their alterations in
neurodegenerative diseases and some cancers. The mechanisms involved in generating this pathology vary,