Exosome biogenesis and uptake. (A) [1] The process of exosome formation begins with the inward budding of the plasma membrane to form primary endocytic vesicles, which fuse together to create early endosomes. [2] Early endosomes mature into late endosomes at which point a second inward budding occurs to form intraluminal vesicles (ILVs). Late endosomes that contain several ILVs are called multivesicular bodies (MVBs). Biogenesis of exosomes can be either ESCRT (endosomal sorting complex required for transport) dependent or independent. ESCRT proteins (including TSG-101) can be divided in four multimeric complexes (ESCRT-0, -I, -II, -III) each with their defined role in vesicle formation. The accessory proteins Bro1/ALG-2-interacting protein X (ALIX) and vacuolar protein sorting (VPS4) ATPase are involved in stabilizing the complex. Syntenin is a multivalent protein that binds the cytosolic domain of syndecan but also directly interacts with ALIX. ESCRT independent exosome biogenesis involves lipids such as ceramide or tetraspanins. ESCRT dependent and independent mechanisms most likely also work together. [3] The last step in exosome biogenesis is their release into extracellular space. This step includes the transport of MVBs to the plasma membrane, followed by their docking and fusion. This process is regulated by proteins involved in cytoskeletal rearrangements and fusion machinery such as the Rab family of GTPases and the SNARE (soluble NSF attachment protein receptors) family proteins. Different Rab proteins have been implicated in vesicular trafficking of which Rab27a/b are best described. The SNARE protein family encompasses more than 60 members, which induce membrane fusion. The best described SNAREs involved in exosome release are VAMP3 and VAMP7. (B) Different uptake mechanisms have been described. [1] Specific uptake through receptors present on the membrane which is dependent on which ligands are expressed on the membrane of the exosomes. [2] Pinocytosis, during which actin-driven membrane ruffling is triggered in the recipient cell. Lamellipodia will form pinocytic cups in which exosomes, bound to the membrane, are “captured”. Once the pinocytic cups close, they are termed pinocytomes, which will shrink to the size of endosomes. [3] Clathrin-mediated endocytosis is a receptor mediated process whereby clathrin-coated vesicles will be formed, followed by invagination of the membrane and fusion to endosomes. Dynamin 2, clathrin and adaptor protein 2 (AP2) are the best characterized proteins involved in this process. Caveolin-dependent endocytosis is similar to clathrin but involves the presence of caveolae, small plasma membrane invaginations rich in caveolin, cholesterol and sphingolipids. [4] Plasma membrane fusion, during which the membrane of the exosome directly merges with the plasma membrane, releasing exosome content into the cytosol. Rab and SNARE family proteins contribute to this process. This figure was created with biorender.com.