Endocytosis of Extracellular Vesicles and Release of Their Cargo from Endosomes

Abstract

Extracellular vesicles (EVs), such as exosomes, can mediate long-distance communication between cells by delivering biomolecular cargo. It is speculated that EVs undergo back-fusion at multivesicular bodies (MVBs) in recipient cells to release their functional cargo. However, direct evidence is lacking. Tracing the cellular uptake of EVs with high resolution as well as acquiring direct evidence for the release of EV cargo is challenging mainly because of technical limitations. Here, we developed an analytical methodology, combining state-of-the-art molecular tools and correlative light and electron microscopy, to identify the intracellular site for EV cargo release. GFP was loaded inside EVs through the expression of GFP-CD63, a fusion of GFP to the cytosolic tail of CD63, in EV producer cells. In addition, we genetically engineered a cell line which expresses anti-GFP fluobody that specifically recognizes the EV cargo (GFP). Incubation of anti-GFP fluobody-expressing cells with GFP-CD63 EVs resulted in the formation of fluobody punctae, designating cytosolic exposure of GFP. Endosomal damage was not observed in EV acceptor cells. Ultrastructural analysis of the underlying structures at GFP/fluobody double-positive punctae demonstrated that EV cargo release occurs from endosomes/lysosomes. Finally, we show that neutralization of endosomal pH and cholesterol accumulation in endosomes leads to blockage of EV cargo exposure. In conclusion, we report that a fraction of internalized EVs fuse with the limiting membrane of endosomes/lysosomes in an acidification-dependent manner, which results in EV cargo exposure to the cell cytosol.

The Protective Effect of Exercise in Neurodegenerative Diseases: The Potential Role of Extracellular Vesicles

“…Whilst some of the uptake mechanisms have been proven with cell biology-based experiments, there is limited evidence for other pathways. For instance, receptor-mediated endocytosis seems to be the most preferred uptake route of EVs, as suggested by several studies [ 157 , 158 , 159 , 160 ] Regardless, the first step in this process is binding between EVs and their target cells, through specific interactions between proteins such as tetraspanins, integrins, cytokines and lipids enriched at the surfaces of the EVs and receptors such as intercellular adhesion molecules embedded on the plasma membranes of the target cells [ 12 , 161 ]. This has been shown to occur for a number of cell types including liver, lung, lymph node, neural and dendritic and intestinal epithelial cells [ 162 , 163 , 164 ].…”

“…The content of EVs, the composition of the membrane bound proteins on their surface and the presence of specific structures on the target cell all play roles in the targeting of EVs to specific recipient cells. Once EVs are taken up by recipient cells, they follow the endocytic pathway where they can back-fuse with the endosomal membrane and release the contents to the cytoplasm [ 157 , 159 ] or fuse with the lysosomes [ 167 ]. Mechanisms governing these processes are poorly understood.…”

Gap junction internalization and processing in vivo: a 3D immuno-electron microscopy study

“…This occurs with enveloped viruses such as vesicular stomatitis virus (Le Blanc et al, 2005) or coronavirus (Grove and Marsh, 2011), resulting in the release of nucleocapsids into the cytoplasm. There is now direct evidence that this also occurs with extracellular vesicles (Joshi et al, 2020), resulting in release of cargo to the cytoplasm.…”

The Wnt Signalling Pathway: A Tailored Target in Cancer

“…EVs are heterogeneous small membrane-bound carriers with complex cargoes released under both physiological and pathological conditions. Almost any cell can release EVs, which act as inter-cellular mediators modifying target cell fate at closed or distant sites [ 128 ].…”

“…Based on the biogenesis, size, content, mechanisms of release and function, three discrete EV subtypes are recognized: microvesicles (MVs), exosomes, and apoptotic bodies [ 128 ].…”