In vitro fusion of tissue-derived endosomes and lysosomes

We investigated the in vitro fusion of different endocytic compartments der ived from perfused rat liver, where the cells are assumed to be in their ph ysiological state. Specifically labelled early, late and transcytotic endos omes, as well as lysosomes were tested for their fusion properties. In addi tion to the expected ATP-dependent fusion between early endosomes, we obser ved fusion between early and late endosomes with similar efficiency kinetic s and cytosol dependence. Fusion between early endosomes and transcytotic v esicles could not be detected, prolonged incubation of complementary labell ed early endosomes under fusion-supporting conditions followed by Percoll g radient centrifugation revealed the occurrence of fusion product at a dense position, indicating fusion events between tight and dense compartments. I ncubation 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 foll owed by fusion with lysosomes. This was verified by colocalization of fluor escently labelled endosomes and lysosomes, as assessed by laser scanning mi croscopy. 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 ear ly and late endosomes of rat liver exhibit a high fusion competence in vitr o, promoting not only homotypic, but also heterotypic fusion.