Endocytosis: The process of exosome formation begins with the invagination of the cell’s plasma membrane, leading to the formation of early endosomes. This occurs through endocytosis, where the cell engulfs extracellular material by wrapping the plasma membrane around it

Multivesicular Bodies Formation (MVBs): Early endosomes mature into late endosomes, also known as multivesicular bodies (MVBs). The maturation process involves the inward budding of the endosomal membrane, resulting in the formation of intraluminal vesicles (ILVs) within the MVBs.

Cargo Sorting; The selection of proteins, lipids and nucleic acids is a step during exosome biogenesis. These components are selectively incorporated into ILVs while MVBs are being formed. The sorting of cargo into ILVs can be influenced by various mechanisms, including the endosomal sorting complex required for transport (ESCRT) machinery and ESCRT-independent pathways.

ESCRT-Dependent Pathway: The ESCRT machinery comprises protein complexes (ESCRT 0, I, II and III) that play roles in both cargo sorting and ILV formation. ESCRT 0 has the ability to identify cargo. ESCRT I and ESCRT II assist, in reshaping the membrane while

ESCRT III facilitates the separation of vesicles (ILVs).

ESCRT-Independent Pathway: Apart from the pathway that relies on ESCRT there are mechanisms that don’t require ESCRT involvement. These alternative pathways involve lipid rafts, tetraspanins and various molecules that play a role in packaging cargo into exosomes.

MVB Trafficking: Mature MVBs containing ILVs can follow different trafficking pathways. They can either fuse with lysosomes, causing the degradation of their content, or they can move towards the cell membrane for the release of exosomes.

Exosome Release: The fusion of MVBs with the cell membrane results in the release of exosomes into the extracellular space. This process allows the contents of the ILVs, including proteins, lipids, and nucleic acids, to be released and potentially taken up by neighbouring or distant cells.

It’s important to note that while these general steps describe the biogenesis of exosomes, the process can vary depending on cell type and physiological context