title: Dense core lysosomes can fuse with late endosomes and are re-formed from the resultant hybrid organelles.
abstract: Electron microscopy was used to evaluate the function and formation of dense core lysosomes. Lysosomes were preloaded with bovine serum albumin (BSA)-gold conjugates by fluid phase endocytosis using a pulse-chase protocol. The gold particles present in dense core lysosomes and late endosomes were flocculated, consistent with proteolytic degradation of the BSA. A second pulse of BSA-gold also accumulated in the pre-loaded dense core lysosomes at 37 degrees C, but accumulation was reversibly blocked by incubation at 20 degrees C. Time course experiments indicated that mixing of the two BSA-gold conjugates initially occurred upon fusion of mannose 6-phosphate receptor-positive/lysosomal glycoprotein-positive late endosomes with dense core lysosomes. Treatment for 5 hours with wortmannin, a phosphatidyl inositide 3-kinase inhibitor, caused a reduction in number of dense core lysosomes preloaded with BSA-gold and prevented a second pulse of BSA-gold accumulating in them. After wortmannin treatment the two BSA-gold conjugates were mixed in swollen late endosomal structures. Incubation of NRK cells with 0.03 M sucrose resulted in the formation of swollen sucrosomes which were morphologically distinct from preloaded dense core lysosomes and were identified as late endosomes and hybrid endosome-lysosome structures. Subsequent endocytosis of invertase resulted in digestion of the sucrose and re-formation of dense core lysosomes. These observations suggest that dense core lysosomes are biologically active storage granules of lysosomal proteases which can fuse with late endosomes and be re-formed from the resultant hybrid organelles prior to subsequent cycles of fusion and re-formation.
authors.affiliation: Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, UK.
authors.forename: N A
authors.initials: NA
authors.lastname: Bright
authors.forename: B J
authors.initials: BJ
authors.lastname: Reaves
authors.forename: B M
authors.initials: BM
authors.lastname: Mullock
authors.forename: J P
authors.initials: JP
authors.lastname: Luzio
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D015703:Antigens, CD / Q000032:analysis; D056668:Cathepsin L; D002403:Cathepsins / Q000032:analysis; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D010450:Endopeptidases; D011992:Endosomes / Q000737:chemistry / Q000502:physiology* / Q000648:ultrastructure; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000032:analysis; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology / Q000648:ultrastructure; D006026:Glycoside Hydrolases / Q000493:pharmacokinetics; D006046:Gold / Q000493:pharmacokinetics; D006867:Hydrolases / Q000378:metabolism; D007668:Kidney / Q000166:cytology; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000737:chemistry / Q000502:physiology* / Q000648:ultrastructure; D008562:Membrane Glycoproteins / Q000032:analysis; D016253:Microscopy, Immunoelectron; D051381:Rats; D017527:Receptor, IGF Type 2 / Q000032:analysis; D012710:Serum Albumin, Bovine / Q000493:pharmacokinetics; D013395:Sucrose / Q000493:pharmacokinetics; D000077191:Wortmannin; D043324:beta-Fructofuranosidase
chemical_list: D000730:Androstadienes; D015703:Antigens, CD; D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D017527:Receptor, IGF Type 2; D012710:Serum Albumin, Bovine; D013395:Sucrose; D006046:Gold; D006867:Hydrolases; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D056668:Cathepsin L; C534280:Ctsl protein, rat; D000077191:Wortmannin