## 4 Extracellular Vesicles
### Content and Biogenesis  
Cell type and the physiological or pathological state of the parent cell may influence its EV content [73]. To date, the interest in EVs has grown exponentially due to their abundant presence in body fluids, their wide range of regulatory functions and their capacity as vehicles for DNA, mRNAs, miRNAs, proteins, and lipids involved in intercellular communication (Figure 2) [74]. Figure 1: Extracellular vesicles distinguished by size: apoptotic bodies (800–5000 nm), microvesicles (50–1000 nm) and exosomes (30–100 nm). Created with BioRender.com (accessed on 10 October 2021). Figure 2: Exosomal composition in terms of bioactive molecules: lipids, proteins, and nucleic acids. Created with BioRender.com (accessed on 10 October 2021). The protein content of EVs is often used as a marker to recognize EV sub-populations. Exosomes are rich in endosome-associated proteins (e.g., Rab GTPases, SNAREs, annexins, and flotillin), proteins involved in EV biogenesis (e.g., Alix and Tsg101), transmembrane proteins composed of four domains such as tetraspanins (CD63, CD81, CD9 and CD82), major histocompatibility complex (MHC) molecules and cytosolic proteins such as heat shock proteins [20]. EVs also present a lipidic component. Exosomes are enriched in cholesterol, sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine, as well as proteins associated to lipid rafts such as glycosylphosphatidyl-anchored proteins and flotillin. Nevertheless, the lipid content of microvesicles is less known than exosomes and is primarily involved in their budding from the plasma membrane [75]. Regarding nucleic acids, it was reported that microvesicles and exosomes carry single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), genomic DNA, mitochondrial DNA, and reverse-transcribed complementary DNAs [76; 77]. On the other hand, EVs can deliver mRNA, mRNA fragments, long non-coding RNA (lnRNA), miRNA, ribosomal RNA (rRNA) and fragments of tRNA, vault- and Y-RNA [78]. More importantly, miRNAs may act on the behavior of recipient cells by regulating gene expression, immune response, and additional physiological processes [79; 80]. Exosomes and microvesicles have two different pathways of biogenesis. They are constantly produced by viable cells, while apoptotic bodies are released exclusively during the apoptotic phase of the cell cycle [81]. Whereas the releasing mechanisms of apoptotic bodies from the cell membrane have been widely elucidated, those related to microvesicles have been investigated only recently. Microc vesicles are formed by fusion with the internal side of the plasma membrane and requires several rearrangements within the membrane itself such as reorganization of lipid and protein components and Ca\({}^{2+}\) levels [82; 83]. It has been reported that a reduction in cholesterol in microvesicles may compromise their generation in activated neutrophils [84]. In addition, the small GTPase family Rho and the protein kinase associated with Rho (ROCK) induce the biogenesis of microvesicles in various populations of cancer cells by regulating the activity of cytoskeletal components such as actin [85]. Exosomes are defined as intraluminal vesicles (ILVs) formed by the invagination of the endosomal membrane during the maturation of multivesicular endosomes (MVEs). This process leads to the acquisition of cargo and emission of vesicles in the lumen through particular sorting machineries (Figure 3) [86]. Figure 3: Visualization of MVEs by electron microscopy (scale bar = 1 \(\upmu\)m). Biogenesis process of ILVs by budding into early endosomes. MVEs release exosomes by fusion with the plasma membrane or fuse with lysosomes. ILVs = intraluminal vesicles; MVEs = multivesicular endosomes. Created with BioRender.com (accessed on 10 October 2021). The discovery of the endosomal sorting complex required for transport (ESCRT) machinery was important as it seems to be a fundamental component in several cell types for the generation of MVEs and ILVs [87; 88].