Endocytic Delivery to Lysosomes Mediated by Concurrent Fusion and Kissing Events in Living Cells

In mammalian cells, macromolecules internalized by endocytosis are transported via endosomes for digestion by lysosomal acid hydrolases [1Gruenberg J. The endocytic pathway a mosaic of domains.Nat. Rev. Mol. Cell Biol. 2001; 2: 721-730Crossref PubMed Scopus (565) Google Scholar, 2Mullins C. Bonifacino J.S. The molecular machinery of lysosome biogenesis.Bioessays. 2001; 23: 333-343Crossref PubMed Scopus (165) Google Scholar, 3Luzio J.P. Poupon V. Lindsay M.R. Mullock B.M. Piper R.C. Pryor P.R. Membrane dynamics and the biogenesis of lysosomes.Mol. Membr. Biol. 2003; 20: 141-154Crossref PubMed Scopus (121) Google Scholar]. The mechanism by which endosomes and lysosomes exchange content remains equivocal [2Mullins C. Bonifacino J.S. The molecular machinery of lysosome biogenesis.Bioessays. 2001; 23: 333-343Crossref PubMed Scopus (165) Google Scholar, 3Luzio J.P. Poupon V. Lindsay M.R. Mullock B.M. Piper R.C. Pryor P.R. Membrane dynamics and the biogenesis of lysosomes.Mol. Membr. 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Cell Biol. 2000; 149: 1053-1062Crossref PubMed Scopus (263) Google Scholar, 8Pryor P.R. Mullock B.M. Bright N.A. Lindsay M.R. Gray S.R. Richardson S.C. Stewart A. James D.E. Piper R.C. Luzio J.P. Combinatorial SNARE complexes with VAMP7 or VAMP8 define different late endocytic fusion events.EMBO Rep. 2004; 5: 590-595Crossref PubMed Scopus (177) Google Scholar, 9Alberts B. Johnson A. Lewis J. Raff M. Roberts K. Walter P. Molecular Biology of the Cell. Fourth Edition. Garland Science, New York2002Google Scholar]. However, lysosomes are reusable organelles because they remain accessible to endocytic enzyme replacement therapies [10Desnick R.J. Schuchman E.H. Enzyme replacement and enhancement therapies lessons from lysosomal disorders.Nat. Rev. Genet. 2002; 3: 954-966Crossref PubMed Scopus (229) Google Scholar] and undergo content mixing with late endosomes [5Mullock B.M. Bright N.A. Fearon C.W. Gray S.R. Luzio J.P. Fusion of lysosomes with late endosomes produces a hybrid organelle of intermediate density and is NSF dependent.J. Cell Biol. 1998; 140: 591-601Crossref PubMed Scopus (164) Google Scholar, 6Bright N.A. Reaves B.J. Mullock B.M. Luzio J.P. Dense core lysosomes can fuse with late endosomes and are reformed from the resultant hybrid organelles.J. Cell Sci. 1997; 110: 2027-2040Crossref PubMed Google Scholar]. The maturation model, which proposes that endosomes mature into lysosomes [9Alberts B. Johnson A. Lewis J. Raff M. Roberts K. Walter P. Molecular Biology of the Cell. Fourth Edition. Garland Science, New York2002Google Scholar], cannot explain these observations. Three mechanisms for content mixing have been proposed. The first is vesicular transport, best supported by a yeast cell-free assay [11Vida T. Gerhardt B. A cell-free assay allows reconstitution of vps33p-dependent transport to the yeast vacuole/lysosome.J. Cell Biol. 1999; 146: 85-97Crossref PubMed Google Scholar]. The second suggests that endosomes and lysosomes engage in repeated transient fusions termed "kiss-and-run" [4Storrie B. Desjardins M. The biogenesis of lysosomes is it a kiss and run, continuous fusion and fission process?.Bioessays. 1996; 18: 895-903Crossref PubMed Scopus (119) Google Scholar]. The third is that endosomes and lysosomes fuse completely, yielding hybrid compartments from which lysosomes reform [3Luzio J.P. Poupon V. Lindsay M.R. Mullock B.M. Piper R.C. Pryor P.R. Membrane dynamics and the biogenesis of lysosomes.Mol. Membr. Biol. 2003; 20: 141-154Crossref PubMed Scopus (121) Google Scholar, 5Mullock B.M. Bright N.A. Fearon C.W. Gray S.R. Luzio J.P. Fusion of lysosomes with late endosomes produces a hybrid organelle of intermediate density and is NSF dependent.J. Cell Biol. 1998; 140: 591-601Crossref PubMed Scopus (164) Google Scholar, 6Bright N.A. Reaves B.J. Mullock B.M. Luzio J.P. Dense core lysosomes can fuse with late endosomes and are reformed from the resultant hybrid organelles.J. Cell Sci. 1997; 110: 2027-2040Crossref PubMed Google Scholar, 7Pryor P.R. Mullock B.M. Bright N.A. Gray S.R. Luzio J.P. The role of intraorganellar Ca2+ in late endosome-lysosome heterotypic fusion and in the reformation of lysosomes from hybrid organelles.J. Cell Biol. 2000; 149: 1053-1062Crossref PubMed Scopus (263) Google Scholar, 8Pryor P.R. Mullock B.M. Bright N.A. Lindsay M.R. Gray S.R. Richardson S.C. Stewart A. James D.E. Piper R.C. Luzio J.P. Combinatorial SNARE complexes with VAMP7 or VAMP8 define different late endocytic fusion events.EMBO Rep. 2004; 5: 590-595Crossref PubMed Scopus (177) Google Scholar], termed "fusion-fission" [2Mullins C. Bonifacino J.S. The molecular machinery of lysosome biogenesis.Bioessays. 2001; 23: 333-343Crossref PubMed Scopus (165) Google Scholar]. We utilized time-lapse confocal microscopy to test these hypotheses in living cells. Lysosomes were loaded with rhodamine dextran by pulse-chase, and subsequently late endosomes were loaded with Oregon green 488 dextran. Direct fusions were observed between endosomes and lysosomes, and one such event was captured by correlative electron microscopy. Fluorescence intensity analyses of endosomes that encountered lysosomes revealed a gradual accumulation of lysosomal content. Our data are compatible with a requirement for direct contact between organelles before content is exchanged.