Fig. 5.

Hijacking of endogenous endosomes revealed by electron microscopy. Hippocampal neurons in microfluidic devices in Ch1 according to model 2 were treated with exosomes isolated from mouse brains and labeled with FM1–43FX, a fluorescent dye that reacts with diaminobenzidine (DAB) to form insoluble dark precipitates that are visualized by electron microscopy. a Electron microscopy image of a neuronal soma in Ch1, showing that the majority of endosomes contain DAB-positive exosomes of exogenous origin (black arrowheads). A few endosomes are DAB-negative (white arrowheads). b High magnification of endosomes located in the neuronal soma, showing a mixture of exogenous DAB-positive exosomes (black arrowheads) and endogenous DAB-negative intraluminal nanovesicles (black arrows). c Somatic endosome showing engulfment and fusion with a smaller endosome containing DAB-positive exosomes (black arrowhead “a”). An exogenous DAB-positive exosome can be seen close to the fused endosome (black arrowhead “b”). Endogenous DAB-negative intraluminal nanovesicles are also visualized (black arrows). d-f Endosomes found in axons. (d) Low magnification of axons transporting a small DAB-positive endosome (black arrowhead “a”) and a large endosome in front of it (black arrowhead “b”). e High magnification of a large axonal endosome containing a mixture of DAB-positive (black arrowheads) and DAB-negative vesicles (black arrows). f Axonal termini showing endosome fusion with the plasma membrane (indicated with “!”) during exosome release and potential residue of the back-fusion with the limiting membrane of the endosome (_*). Exogenous DAB-positive (black arrowheads) and endogenous DAB-negative exosomes (black arrows). g-i Endosomes found in dendrites. g Low magnification of a dendrite demonstrating the presence of endosomes carrying both DAB-positive exosomes (black arrowheads) and DAB-negative intraluminal nanovesicles (black arrows). h and i High magnification of dendritic endosomes carrying DAB-positive exosomes less than 100 nm diameter (black arrowheads) together with DAB-negative vesicles of a similar size (black arrows). m, mitochondria; n, nucleus