• Endocytosis of Extracellular Vesicles and Release of Their Cargo from Endosomes.

Endocytosis of Extracellular Vesicles and Release of Their Cargo from Endosomes.

ACS nano (2020-04-14)
Bhagyashree S Joshi, Marit A de Beer, Ben N G Giepmans, Inge S Zuhorn

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.

Product Number
Product Description

Puromycin dihydrochloride from Streptomyces alboniger, powder, BioReagent, suitable for cell culture
Bovine Serum Albumin, heat shock fraction, pH 7, ≥98%
Thiazolyl Blue Tetrazolium Bromide, 98%
DAPI, for nucleic acid staining
Poly-L-lysine hydrobromide, mol wt 30,000-70,000
Sodium cacodylate trihydrate, ≥98%
TWEEN® 20, viscous liquid
Immobilon®-FL PVDF Membrane, 1 roll, 27 cm x 3.75 m, 0.45 µm pore size, Hydrophobic PVDF Transfer Membrane with low background fluorescence for Western blotting. Compatible with visible and infrared fluorescent probes.
U18666A, A cell-permeable, amphiphilic amino-steroid that alters intracellular membrane protein trafficking by impairing intracellular biosynthesis and transport of LDL-derived cholesterol.
ECO TWEEN® 20, viscous liquid