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. 1982 Jun 1;93(3):866–874. doi: 10.1083/jcb.93.3.866

Intralysosomal accumulation of polyanions. I. Fusion of pinocytic and phagocytic vacuoles with secondary lysosomes

PMCID: PMC2112160  PMID: 6181074

Abstract

The long-term exposure of macrophages to low concentrations of a number of polyanions leads to their accumulation in high concentration within secondary lysosomes. This was associated with enlargement of the lysosomes, the presence of membranous whorls, and intense toluidine blue staining of the organelles at pH 1.0. After the ingestion of a particulate load by these cells, newly formed phagocytic vacuoles failed to fuse with polyanion-laden lysosomes. The lack of fusion was evident in both fluorescence and electron micrographic studies which followed the transfer of acridine orange or Thorotrast from 2 degrees lysosomes to phagosomes. Agents that inhibited phagosome-lysosome (P-L) fusion included molecules containing high densities of sulfate, sulfonate, or carboxylate residues. Dextran sulfate (DS) in microgram/ml quantities was an excellent inhibitor, whereas nonsulfated dextran (D) was without effect at 1,000-fold higher concentrations. In contrast to their effects on P-L fusion, polyanions failed to influence the fusion of pinocytic vesicles with 2 degrees lysosomes. The uptake, intravacuolar distribution, and intralysosomal digestion of fluid-phase pinocytic markers were unaltered in lysosomes containing either D or DS. Furthermore, subcellular fractionation studies showed that the fluid-phase pinocytic marker HRP was efficiently transferred from pinosomes to large, dense 2 degrees lysosomes containing DS.

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Selected References

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  1. Anderson R. G., Brown M. S., Goldstein J. L. Role of the coated endocytic vesicle in the uptake of receptor-bound low density lipoprotein in human fibroblasts. Cell. 1977 Mar;10(3):351–364. doi: 10.1016/0092-8674(77)90022-8. [DOI] [PubMed] [Google Scholar]
  2. Basu S. K., Brown M. S., Ho Y. K., Goldstein J. L. Degradation of low density lipoprotein . dextran sulfate complexes associated with deposition of cholesteryl esters in mouse macrophages. J Biol Chem. 1979 Aug 10;254(15):7141–7146. [PubMed] [Google Scholar]
  3. Burstein M., Scholnick H. R., Morfin R. Rapid method for the isolation of lipoproteins from human serum by precipitation with polyanions. J Lipid Res. 1970 Nov;11(6):583–595. [PubMed] [Google Scholar]
  4. CORNWELL D. G., KRUGER F. A. Molecular complexes in the isolation and characterization of plasma lipoproteins. J Lipid Res. 1961 Apr;2:110–134. [PubMed] [Google Scholar]
  5. Constantopoulos G., Rees S., Cragg B. G., Barranger J. A., Brady R. O. Experimental animal model for mucopolysaccharidosis: suramin-induced glycosaminoglycan and sphingolipid accumulation in the rat. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3700–3704. doi: 10.1073/pnas.77.6.3700. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Edelson P. J., Cohn Z. A. 5'-Nucleotidase activity of mouse peritoneal macrophages. I. Synthesis and degradation in resident and inflammatory populations. J Exp Med. 1976 Dec 1;144(6):1581–1595. doi: 10.1084/jem.144.6.1581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Edelson P. J., Cohn Z. A. Effects of concanavalin A on mouse peritoneal macrophages. I. Stimulation of endocytic activity and inhibition of phago-lysosome formation. J Exp Med. 1974 Nov 1;140(5):1364–1386. doi: 10.1084/jem.140.5.1364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Geisow M. J., Beaven G. H., Hart P. D., Young M. R. Site of action of a polyanion inhibitor of phagosome-lysosome fusion in cultured macrophages. Exp Cell Res. 1980 Mar;126(1):159–165. doi: 10.1016/0014-4827(80)90481-4. [DOI] [PubMed] [Google Scholar]
  9. Geisow M. J., D'Arcy Hart P., Young M. R. Temporal changes of lysosome and phagosome pH during phagolysosome formation in macrophages: studies by fluorescence spectroscopy. J Cell Biol. 1981 Jun;89(3):645–652. doi: 10.1083/jcb.89.3.645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Goren M. B., D'Arcy Hart P., Young M. R., Armstrong J. A. Prevention of phagosome-lysosome fusion in cultured macrophages by sulfatides of Mycobacterium tuberculosis. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2510–2514. doi: 10.1073/pnas.73.7.2510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Graham R. C., Jr, Karnovsky M. J. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J Histochem Cytochem. 1966 Apr;14(4):291–302. doi: 10.1177/14.4.291. [DOI] [PubMed] [Google Scholar]
  12. Hart P. D., Young M. R. Interference with normal phagosome-lysosome fusion in macrophages, using ingested yeast cells and suramin. Nature. 1975 Jul 3;256(5512):47–49. doi: 10.1038/256047a0. [DOI] [PubMed] [Google Scholar]
  13. Hart P. D., Young M. R. The effect of inhibitors and enhancers of phagosome--lysosome fusion in cultured macrophages on the phagosome membranes of ingested yeasts. Exp Cell Res. 1979 Feb;118(2):365–375. doi: 10.1016/0014-4827(79)90160-5. [DOI] [PubMed] [Google Scholar]
  14. Kielian M. C., Cohn Z. A. Modulation of phagosome-lysosome fusion in mouse macrophages. J Exp Med. 1981 Apr 1;153(4):1015–1020. doi: 10.1084/jem.153.4.1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kielian M. C., Cohn Z. A. Phagosome-lysosome fusion. Characterization of intracellular membrane fusion in mouse macrophages. J Cell Biol. 1980 Jun;85(3):754–765. doi: 10.1083/jcb.85.3.754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kielian M. C., Cohn Z. A. Phorbol myristate acetate stimulates phagosome-lysosome fusion in mouse macrophages. J Exp Med. 1981 Jul 1;154(1):101–111. doi: 10.1084/jem.154.1.101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  18. Mahoney E. M., Hamill A. L., Scott W. A., Cohn Z. A. Response of endocytosis to altered fatty acyl composition of macrophage phospholipids. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4895–4899. doi: 10.1073/pnas.74.11.4895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mellman I. S., Steinman R. M., Unkeless J. C., Cohn Z. A. Selective iodination and polypeptide composition of pinocytic vesicles. J Cell Biol. 1980 Sep;86(3):712–722. doi: 10.1083/jcb.86.3.712. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Muller W. A., Steinman R. M., Cohn Z. A. The membrane proteins of the vacuolar system I. Analysis of a novel method of intralysosomal iodination. J Cell Biol. 1980 Jul;86(1):292–303. doi: 10.1083/jcb.86.1.292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Munthe-Kaas A. C., Seglen P. O. The use of Metrizamide as a gradient medium for isopycnic separation of rat liver cells. FEBS Lett. 1974 Aug 1;43(3):252–256. doi: 10.1016/0014-5793(74)80654-x. [DOI] [PubMed] [Google Scholar]
  22. Nichols B. A. Normal rabbit alveolar macrophages. I. The phagocytosis of tubular myelin. J Exp Med. 1976 Oct 1;144(4):906–919. doi: 10.1084/jem.144.4.906. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nishida T., Cogan U. Nature of the interaction of dextran sulfate with low density lipoproteins of plasma. J Biol Chem. 1970 Sep 25;245(18):4689–4697. [PubMed] [Google Scholar]
  24. Pesanti E. L. Suramin effects on macrophage phagolysosome formation and antimicrobial activity. Infect Immun. 1978 May;20(2):503–511. doi: 10.1128/iai.20.2.503-511.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Peters T. J., Müller M., De Duve C. Lysosomes of the arterial wall. I. Isolation and subcellular fractionation of cells from normal rabbit aorta. J Exp Med. 1972 Nov 1;136(5):1117–1139. doi: 10.1084/jem.136.5.1117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Poste G., Allison A. C. Membrane fusion. Biochim Biophys Acta. 1973 Dec 28;300(4):421–465. doi: 10.1016/0304-4157(73)90015-4. [DOI] [PubMed] [Google Scholar]
  27. Silverstein S. C., Steinman R. M., Cohn Z. A. Endocytosis. Annu Rev Biochem. 1977;46:669–722. doi: 10.1146/annurev.bi.46.070177.003321. [DOI] [PubMed] [Google Scholar]
  28. Steinman R. M., Brodie S. E., Cohn Z. A. Membrane flow during pinocytosis. A stereologic analysis. J Cell Biol. 1976 Mar;68(3):665–687. doi: 10.1083/jcb.68.3.665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Steinman R. M., Cohn Z. A. The interaction of soluble horseradish peroxidase with mouse peritoneal macrophages in vitro. J Cell Biol. 1972 Oct;55(1):186–204. doi: 10.1083/jcb.55.1.186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Terho T. T., Hartiala K. Method for determination of the sulfate content of glycosaminoglycans. Anal Biochem. 1971 Jun;41(2):471–476. doi: 10.1016/0003-2697(71)90167-9. [DOI] [PubMed] [Google Scholar]

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