Phagosome-lysosome fusion: Characterization of intracellular membrane fusion in mouse macrophages

Several approaches have been used to study the determinants of phagosomelysosome fusion in intact mouse macrophages. Lysosomes were labeled with the fluorescent vital dye acridine orange and the rate and extent of their fusion with yeast-containing phagosomes was monitored by fluorescence microscopy. Fusion was also assayed by electron microscopy, using horseradish peroxidase or thorium dioxide as a marker for secondary lysosomes. Good agreement was found with results obtained from vitally stained cells, thin-section samples with an enzymatic marker, and thorium dioxide-labeled samples evaluated by stereology. The rate of fusion as assayed by fluorescence was not affected by the number of particles ingested, serum concentration, or prior uptake of digestible or nondigestible substances. With this assay it was possible to observe the rate of fusion separate from and uninfluenced by the phagocytic rate. Both the rate and extent of fusion were dramatically increased after several days in culture and similar changes were found by use of the EM assays. Fusion was strongly affected by incubation temperature, having a Q₁₀ of 2.5. No detectable fusion occurred below 15°C, and this inhibition was rapidly reversed when cells were returned to 37°C.