Fig. 2. Biogenesis and identification of exosomes.

Fluid and extracellular constituents such as proteins, lipids, metabolites, small molecules, and ions can enter cells, along with cell surface proteins, through endocytosis and plasma membrane invagination. The resulting plasma membrane bud formation in the luminal side of the cell presents with outside-in plasma membrane orientation. This budding process leads to the formation of ESEs or possible fusion of the bud with ESEs preformed by the constituents of the endoplasmic reticulum (ER), trans-Golgi network (TGN), and mitochondria. The ESEs could also fuse with the ER and TGN, possibly explaining how the endocytic cargo reaches them. Some of the ESEs can therefore contain membrane and luminal constituents that can represent diverse origins. ESEs give rise to LSEs. Second invagination in the LSE leads to the generation of ILVs, and this step can lead to further modification of the cargo of the future exosomes, with cytoplasmic constituents entering the newly forming ILV. As part of the formation of ILVs, proteins (that were originally on the cell surface) could be distinctly distributed among ILVs. Depending on the invagination volume, the process could give rise to ILVs of different sizes with distinct content. LSEs give rise to MVBs with defined collection of ILVs (future exosomes). MVBs can fuse with autophagosomes, and ultimately the contents can undergo degradation in the lysosomes. The degradation products could be recycled by the cells. MVBs can also directly fuse with lysosomes for degradation. MVBs that do not follow this trajectory can be transported to the plasma membrane through the cytoskeletal and microtubule network of the cell and dock on the luminal side of the plasma membrane with the help of MVB-docking proteins. Exocytosis follows and results in the release of the exosomes with a similar lipid bilayer orientation as the plasma membrane. Several proteins are implicated in exosome biogenesis and include Rab GTPases, ESCRT proteins (see text), as well as others that are also used as markers for exosomes (CD9, CD81, CD63, flotillin, TSG101, ceramide, and Alix). Exosome surface proteins include tetraspanins, integrins, immunomodulatory proteins, and more. Exosomes can contain different types of cell surface proteins, intracellular protein, RNA, DNA, amino acids, and metabolites.