Mechanisms of Multivesicular Body Biogenesis and Exosome Release

Exosomes are small vesicles with a diameter of approximately 50-100 nm that are secreted by a number of different cells and function in a multitude of intercellular signaling processes. Exosomes are thought to derive from release of intraluminal vesicles (ILVs) in multivesicular bodies (MVBs) after the fusion of MVBs with the plasma membrane.To understand the little explored molecular details of how cargoes are sorted to these subsets of ILVs in MVBs and secreted with exosomes, the transport of proteolipid protein (PLP) in Oli-neu cells, an oligodendroglial precursor cell line, was studied. We found that PLP was transported to distinct subdomains of the endosomal membrane and that the transfer of PLP into the lumen of endosomes did not depend on the function of the endosomal sorting complex required for transport (ESCRT) machinery but required ceramide formation. The purified exosomes were enriched with ceramide and inhibition of neutral sphingomyelinase activity reduced the release of exosomes. Sphingomyelin hydrolysis on the outer leaflet of phase separated giant unilamellar vesicles resulted in intraluminal budding from the lipid ordered phase. These results identified a novel lipid-dependent pathway for cargo transport to the ILVs in MVBs for exosomes formation.Rab family guanosine triphosphatases (GTPases) are regulators involved in intracellular membrane traffcking. A screen for the effects of Rab GTPase activating proteins (GAPs) on exosome release was performed. Expression of TBC1D10B, RN-tre, TBC1D10A, TBC1D10C and TBC1D15 reduced exosome secretion in a catalytic activity dependent manner. Rab35 was identified to be the target of TBC1D10A-C. Inhibition of Rab35 function impaired exosome secretion, induced accumulation of PLP containing endosomes and increased the mobility of endosomes closed to the plasma membrane, suggesting a function in MVB tethering. Furthermore, Rab35 was biochemically purified together with non-compact myelin, where MVBs were observed, providing a basis for understanding the biogenesis and functions of exosomes in the central nervous system.