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.

Keywords: cargo delivery; correlative microscopy; endosomal escape; endosomes; extracellular vesicles; nanobody.

Figure 1

Experimental setup to elucidate the intracellular site of EV-cargo release. EVs interacting with recipient cells can release their cargo via: (i) direct fusion with the plasma membrane; (ii) kiss and run fusion with the endoplasmic reticulum; (iii) fusion with the endosome membrane; and (iv) endosomal rupture. (A) In cells engineered to cytosolically express monomeric azami-green galectin-3 fusion protein (mAG-gal3), mAG-gal3 punctae formation only occurs in case of endosomal rupture (iv). (B) In cells engineered to cytosolically express mCherry-tagged anti-GFP fluobody, mCherry punctae formation only occurs in case of fusion of GFP-CD63 EVs (i.e., membrane-bound GFP inside the EV) with the plasma membrane (i) or endosome membrane (iii).

Figure 2

Exogenously added EVs localize in membrane-bound compartments in HEK293T acceptor cells. (A) HEK293T cells incubated for 12 h with GFP-CD63 EVs show a punctate staining pattern in the cytosol (scale bars, 10 μm). (B) Correlative light (green) and EM (greyscale) microscopy for ultrastructural analysis of the internalized EVs (GFP punctae) of the boxed area in (A) (scale bars, 5 μm). (C) Underlying ultrastructures of areas 1–3 in (B) reveal vesicular structures. Additional snap shots available in Supplementary Figure 3B. (Scale bars, 0.2 μm.) (D) Structures given in (C) are labeled with QDs (indicated by arrowhead) following anti-GFP immunolabeling, confirming the presence of GFP-CD63 EVs within the vesicular structures (scale bars, 0.2 μm). All EM data sets at full resolution are available viawww.nanotomy.org.

Figure 3

EVs do not induce endosomal permeabilization. (A) Cartoon illustrating the galectin-3-based assay to detect endosomal permeabilization. HEK293T cells are engineered to cytosolically express monomeric azami-green galectin-3 fusion protein (mAG-gal3). Upon endosomal permeabilization, galactoside residues at the inner leaflet of the endosome are accessible to mAG-gal3, resulting in mAG-gal3 accumulation in the endosome and puncta formation. (B) Fluorescence images of mAG-gal3 expressing HEK293T cells untreated (control), treated with CD63-RFP EVs (EV) and transfected with Lipofectamine (Lipo) (t = 12 h). Red, EVs; green, mAG-gal3; blue, nucleus (scale bars, 10 μm). (C) Quantification of CD63-RFP EV uptake (red line) and mAG-gal3 punctae formation (green line) in cells over time (error bars represent SD, n = 3, ≥ 36 cells analyzed per time point). (D) Quantification of mAG-gal3 punctae upon 12 h treatment of mAG-Gal3 HEK293T cells with EVs and Lipofectamine-based lipoplexes. mAG-gal3 punctae only appear in cells with lipoplex treatment (n = 3; ≥ 45 cells analyzed per condition; **P < 0.01, ***P < 0.001, ns; not significant, ANOVA Tukey’s post hoc test).

Figure 4

EV cargo release occurs from endosomes. (A) Cartoon illustrating the experimental design to identify EV cargo exposure to the cell cytosol. When GFP-CD63 EVs in endosomes undergo fusion with the endosome membrane in anti-GFP fluobody (mCherry) HEK293T cells, the anti-GFP fluobody can access and recognize the GFP at the EV interior, resulting in formation of mCherry punctae. (B) Fluorescence images of anti-GFP fluobody (mCherry) HEK293T cells incubated with GFP-CD63 EVs for 4, 8, and 12 h. Yellow punctae represent colocalization. Green, EV; red, fluobody (scale bars, 10 μm). (C) EV uptake (green line) and colocalization with fluobody punctae (yellow line). Colocalization of GFP and mCherry indicate EV cargo exposure to the cell cytosol (error bars represent SD, n = 3, ≥ 36 cells analyzed per time point). (D) Correlative light (red + green) and EM (greyscale) microscopy of anti-GFP fluobody (mCherry) cells incubated with GFP-CD63 EVs for 12 h. Numbers 1–4 indicate areas of red and green (yellow) colocalization (scale bars, 2 μm). (E) The underlying ultrastructure of intracellular sites of EV cargo exposure to the cell cytosol (areas 1–4 in D). Membrane-bound structures containing numerous ILVs (1, 3, and 4) represent MVBs, while structures with electron-dense interior (2) represent lysosomes (scale bars, 0.2 μm). Complete data set at maximum resolution is available at www.nanotomy.org. (F) Immunostaining for the late endosome/lysosome marker LAMP1 in anti-GFP fluobody cells incubated for 12 h with GFP-CD63 EVs. Green, EV; red, fluobody; 633, antibody staining color-coded blue (scale bars, 10 μm).

Figure 5

EV cargo release is blocked by treatment of acceptor cells with the V-ATPase inhibitor Bafilomycin A1 or U18666A, an inhibitor of cholesterol export from late endosomes/lysosomes. (A) Anti-GFP fluobody (mCherry) expressing cells were incubated for 12 h with GFP-CD63 EVs in the absence and presence of BafA1 or U18666A. Note that colocalization (yellow) between EVs (green) and fluobody punctae (red) is largely absent in BafA1 and U18666A treated cells. Green, EV; red, fluobody; blue, nucleus (scale bars, 10 μm). (B) Quantification of colocalization of GFP-CD63 EVs and fluobody punctae after 12 h incubation in the absence and presence of BafA1 or U18666A. Number of GFP/mCherry double-positive spots in control cells is set at 100% (error bars indicate SD, n = 3, ≥ 24 cells analyzed per condition; *P < 0.05, **P < 0.001, two-tailed Student’s t-test).