HOMOTYPIC LYSOSOME FUSION IN MACROPHAGES - ANALYSIS USING AN IN-VITROASSAY

Lysosomes are dynamic structures capable of fusing with endosomes as w ell as other lysosomes. We examined the biochemical requirements for h omotypic lysosome fusion in vitro using lysosomes obtained from rabbit alveolar macrophages or the cultured macrophage-like cell line, J774E . The in vitro assay measures the formation of a biotinylated HRP-avid in conjugate, in which biotinylated HRP and avidin were accumulated in lysosomes by receptor-mediated endocytosis. We determined that lysoso me fusion in vitro was time- and temperature-dependent and required AT P and an N-ethylmaleimide (NEM)-sensitive factor from cytosol. The NEM -sensitive factor was NSF as purified recombinant NSF could completely replace cytosol in the fusion assay whereas a dominant-negative mutan t NSF inhibited fusion. Fusion in vitro was extensive; up to 30% of pu rified macrophage lysosomes were capable of self-fusion. Addition of G TP gamma s to the in vitro assay inhibited fusion in a concentration-d ependent manner. Purified GDP-dissociation inhibitor inhibited homotyp ic lysosome fusion suggesting the involvement of rabs. Fusion was also inhibited by the heterotrimeric G protein activator mastoparan, but n ot by its inactive analogue Mas-17. Pertussis toxin, a G alpha(i) acti vator, inhibited in vitro lysosome fusion whereas cholera toxin, a G a lpha(s) activator did not inhibit the fusion reaction. Addition of age nts that either promoted or disrupted microtubule function had little effect on either the extent or rate of lysosome fusion. The high value of homotypic fusion was supported by in vivo experiments examining ly sosome fusion in heterokaryons formed between cells containing fluores cently labeled lysosomes. In both macrophages and J774E cells, almost complete mixing of the lysosome labels was observed within 1-3 h of UV sendai-mediated cell fusion. These studies provide a model system for identifying the components required for lysosome fusion.