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© The Rockefeller University Press, 0021-9525/1997//665 $5.00
The Journal of Cell Biology, Volume 139, Number 3, , 1997 665-673

Article

Homotypic Lysosome Fusion in Macrophages: Analysis Using an In Vitro Assay



Diane M. Ward, Jonathan D. Leslie, and Jerry Kaplan

Department of Pathology, Division of Cell Biology and Immunology, University of Utah Health Science Center, Salt Lake City, Utah 84132

Lysosomes are dynamic structures capable of fusing with endosomes as well as other lysosomes. We examined the biochemical requirements for homotypic 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–avidin conjugate, in which biotinylated HRP and avidin were accumulated in lysosomes by receptor-mediated endocytosis. We determined that lysosome fusion in vitro was time- and temperature-dependent and required ATP 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 mutant NSF inhibited fusion. Fusion in vitro was extensive; up to 30% of purified macrophage lysosomes were capable of self-fusion. Addition of GTP{gamma}s to the in vitro assay inhibited fusion in a concentration-dependent manner. Purified GDP-dissociation inhibitor inhibited homotypic lysosome fusion suggesting the involvement of rabs. Fusion was also inhibited by the heterotrimeric G protein activator mastoparan, but not by its inactive analogue Mas-17. Pertussis toxin, a G{alpha}i activator, inhibited in vitro lysosome fusion whereas cholera toxin, a G{alpha}s activator did not inhibit the fusion reaction. Addition of agents 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 lysosome fusion in heterokaryons formed between cells containing fluorescently 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.

Abbreviations used in this paper: ARF, ADP ribosylation factor; b-HRP, biotinylated horseradish peroxidase; b-insulin, biotinylated insulin; Chtx, Cholera toxin; GDI, guanine nucleotide–dissociation inhibitor; HB, homogenization buffer; HMEM, Hanks' minimal essential medium; 125I-Tf(Fe)2, 125I-transferrin; {alpha}M125I-T, {alpha}macroglobulin-125I-trypsin; PNS, postnuclear supernatant; Ptx, Pertussis toxin.

Address all correspondence to J. Kaplan, Department of Pathology, Division of Cell Biology and Immunology, University of Utah Health Science Center, Salt Lake City, UT 84132. Tel.: (801) 581-7427. Fax: (801) 581-4517. E-mail: kaplan{at}bioscience.bilogy.utah.edu


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