In vitro fusion of tissue-derived endosomes and lysosomes

We investigated the in vitro fusion of different endocytic compartments derived from perfused rat liver, where the cells are assumed to be in their physiological state. Specifically labelled early, late and transcytotic endosomes, as well as lysosomes were tested for their fusion properties. In addition to the expected ATP-dependent fusion between early endosomes, we observed fusion between early and late endosomes with similar efficiency, kinetics and cytosol dependence. Fusion between early endosomes and transcytotic vesicles could not be detected. Prolonged incubation of complementary labelled early endosomes under fusion-supporting conditions followed by Percoll gradient centrifugation revealed the occurrence of fusion product at a dense position, indicating fusion events between light and dense compartments. Incubation of membrane preparations containing avidin-labelled endosomes and biotin-dextran-loaded lysosomes resulted in the formation of avidin-biotin complexes, indicating that fusion between early and late endosomes is followed by fusion with lysosomes. This was verified by colocalization of fluorescently labelled endosomes and lysosomes, as assessed by laser scanning microscopy. Endosome fusion, as well as content mixing between endosomes and lysosomes, were dependent on temperature and ATP, and could be inhibited by N-ethylmaleimide (NEM). The NEM-sensitivity was localised on endosomes and in the cytosol, but not on lysosomes. These observations indicate that early and late endosomes of rat liver exhibit a high fusion competence in vitro, promoting not only homotypic, but also heterotypic fusion.