Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1990 Apr 1;110(4):1013–1022. doi: 10.1083/jcb.110.4.1013

Fusion of sequentially internalized vesicles in alveolar macrophages

PMCID: PMC2116061  PMID: 1691186

Abstract

Previously we reported that internalized ligand-receptor complexes are transported within the alveolar macrophage at a rate that is independent of the ligand and/or receptor but is dependent on the endocytic apparatus (Ward, D. M., R. S. Ajioka, and J. Kaplan. 1989. J. Biol. Chem. 264:8164-8170). To probe the mechanism of intracellular vesicle transport, we examined the ability of vesicles internalized at different times to fuse. The mixing of ligands internalized at different times was studied using the 3,3'-diaminobenzidine/horseradish peroxidase density shift technique. The ability of internalized vesicles to fuse was dependent upon their location in the endocytic pathway. When ligands were administered as tandem pulses a significant amount of mixing (20-40%) of vesicular contents was observed. The pattern of mixing was independent of the ligands employed (transferrin, mannosylated BSA, or alpha macroglobulin), the order of ligand addition, and temperature (37 degrees C or 28 degrees C). Fusion was restricted to a brief period immediately after internalization. The amount of fusion in early endosomes did not increase when cells, given tandem pulses, were chased such that the ligands further traversed the early endocytic pathway. Little fusion, also, was seen when a chase was interposed between the two ligand pulses. The temporal segregation of vesicle contents seen in early endosomes was lost within late endosomes. Extensive mixing of vesicle contents was observed in the later portion of the endocytic pathway. This portion of the pathway is defined by the absence of internalized transferrin and is composed of ligands en route to lysosomes. Incubation of cells in iso-osmotic medium in which Na+ was replaced by K+ inhibited movement of internalized ligands to the lysosome, resulting in ligand accumulation within the late endocytic pathway. The accumulation of ligand was correlated with extensive mixing of sequentially internalized ligands. Although significant amounts of ligand degradation were observed, this compartment was devoid of conventional lysosomal markers such as acid glycosidases. These results indicate changing patterns of vesicle fusion within the endocytic pathway, with a complete loss of temporal ligand segregation in a prelysosomal compartment.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ajioka R. S., Kaplan J. Characterization of endocytic compartments using the horseradish peroxidase-diaminobenzidine density shift technique. J Cell Biol. 1987 Jan;104(1):77–85. doi: 10.1083/jcb.104.1.77. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ajioka R. S., Kaplan J. Intracellular pools of transferrin receptors result from constitutive internalization of unoccupied receptors. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6445–6449. doi: 10.1073/pnas.83.17.6445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baenziger J. U., Fiete D. Recycling of the hepatocyte asialoglycoprotein receptor does not require delivery of ligand to lysosomes. J Biol Chem. 1982 Jun 10;257(11):6007–6009. [PubMed] [Google Scholar]
  4. Baenziger J. U., Fiete D. Separation of two populations of endocytic vesicles involved in receptor-ligand sorting in rat hepatocytes. J Biol Chem. 1986 Jun 5;261(16):7445–7454. [PubMed] [Google Scholar]
  5. Braell W. A. Fusion between endocytic vesicles in a cell-free system. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1137–1141. doi: 10.1073/pnas.84.5.1137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chang T. M., Kullberg D. W. Diacytosis of 125I-asialoorosomucoid by rat hepatocytes. A non-lysosomal pathway insensitive to inhibition by inhibitors of ligand degradation. Biochim Biophys Acta. 1984 Nov 13;805(3):268–276. doi: 10.1016/0167-4889(84)90082-x. [DOI] [PubMed] [Google Scholar]
  7. Courtoy P. J., Quintart J., Baudhuin P. Shift of equilibrium density induced by 3,3'-diaminobenzidine cytochemistry: a new procedure for the analysis and purification of peroxidase-containing organelles. J Cell Biol. 1984 Mar;98(3):870–876. doi: 10.1083/jcb.98.3.870. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Davey J., Hurtley S. M., Warren G. Reconstitution of an endocytic fusion event in a cell-free system. Cell. 1985 Dec;43(3 Pt 2):643–652. doi: 10.1016/0092-8674(85)90236-3. [DOI] [PubMed] [Google Scholar]
  9. Davis C. G., Goldstein J. L., Südhof T. C., Anderson R. G., Russell D. W., Brown M. S. Acid-dependent ligand dissociation and recycling of LDL receptor mediated by growth factor homology region. Nature. 1987 Apr 23;326(6115):760–765. doi: 10.1038/326760a0. [DOI] [PubMed] [Google Scholar]
  10. Davoust J., Gruenberg J., Howell K. E. Two threshold values of low pH block endocytosis at different stages. EMBO J. 1987 Dec 1;6(12):3601–3609. doi: 10.1002/j.1460-2075.1987.tb02691.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Diaz R., Mayorga L., Stahl P. In vitro fusion of endosomes following receptor-mediated endocytosis. J Biol Chem. 1988 May 5;263(13):6093–6100. [PubMed] [Google Scholar]
  12. Diment S., Stahl P. Macrophage endosomes contain proteases which degrade endocytosed protein ligands. J Biol Chem. 1985 Dec 5;260(28):15311–15317. [PubMed] [Google Scholar]
  13. Edelson P. J., Erbs C. Plasma membrane localization and metabolism of alkaline phosphodiesterase I in mouse peritoneal macrophages. J Exp Med. 1978 Jan 1;147(1):77–86. doi: 10.1084/jem.147.1.77. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Goldberg R. I., Smith R. M., Jarett L. Insulin and alpha 2-macroglobulin-methylamine undergo endocytosis by different mechanisms in rat adipocytes: I. Comparison of cell surface events. J Cell Physiol. 1987 Nov;133(2):203–212. doi: 10.1002/jcp.1041330202. [DOI] [PubMed] [Google Scholar]
  15. Gorman R. M., Poretz R. D. Resolution of multiple endosomal compartments associated with the internalization of epidermal growth factor and transferrin. J Cell Physiol. 1987 May;131(2):158–164. doi: 10.1002/jcp.1041310204. [DOI] [PubMed] [Google Scholar]
  16. Greenspan P., St Clair R. W. Retroendocytosis of low density lipoprotein. Effect of lysosomal inhibitors on the release of undegraded 125I-low density lipoprotein of altered composition from skin fibroblasts in culture. J Biol Chem. 1984 Feb 10;259(3):1703–1713. [PubMed] [Google Scholar]
  17. Gruenberg J., Griffiths G., Howell K. E. Characterization of the early endosome and putative endocytic carrier vesicles in vivo and with an assay of vesicle fusion in vitro. J Cell Biol. 1989 Apr;108(4):1301–1316. doi: 10.1083/jcb.108.4.1301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kaplan J. Evidence for reutilization of surface receptors for alpha-macroglobulin.protease complexes in rabbit alveolar macrophages. Cell. 1980 Jan;19(1):197–205. doi: 10.1016/0092-8674(80)90401-8. [DOI] [PubMed] [Google Scholar]
  19. Kaplan J., Keogh E. A. Temperature shifts induce the selective loss of alveolar-macrophage plasma membrane components. J Cell Biol. 1982 Jul;94(1):12–19. doi: 10.1083/jcb.94.1.12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kaplan J., Nielsen M. L. Analysis of macrophage surface receptors. I. Binding of alpha-macroglobulin . protease complexes to rabbit alveolar macrophages. J Biol Chem. 1979 Aug 10;254(15):7323–7328. [PubMed] [Google Scholar]
  21. Kaplan J., Nielsen M. L. Analysis of macrophage surface receptors. II. Internalization of alpha-macroglobulin . trypsin complexes by rabbit alveolar macrophages. J Biol Chem. 1979 Aug 10;254(15):7329–7335. [PubMed] [Google Scholar]
  22. Kaplan J., Nielsen M. Pinocytic activity of rabbit alveolar macrophages in vitro. J Reticuloendothel Soc. 1978 Dec;24(6):673–685. [PubMed] [Google Scholar]
  23. 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]
  24. Linderman J. J., Lauffenburger D. A. Analysis of intracellular receptor/ligand sorting in endosomes. J Theor Biol. 1988 May 21;132(2):203–245. doi: 10.1016/s0022-5193(88)80157-7. [DOI] [PubMed] [Google Scholar]
  25. MYRVIK Q. N., LEAKE E. S., FARISS B. Lysozyme content of alveolar and peritoneal macrophages from the rabbit. J Immunol. 1961 Feb;86:133–136. [PubMed] [Google Scholar]
  26. Marshall S. Dual pathways for the intracellular processing of insulin. Relationship between retroendocytosis of intact hormone and the recycling of insulin receptors. J Biol Chem. 1985 Nov 5;260(25):13524–13531. [PubMed] [Google Scholar]
  27. McKinley D. N., Wiley H. S. Reassessment of fluid-phase endocytosis and diacytosis in monolayer cultures of human fibroblasts. J Cell Physiol. 1988 Sep;136(3):389–397. doi: 10.1002/jcp.1041360302. [DOI] [PubMed] [Google Scholar]
  28. Mueller S. C., Hubbard A. L. Receptor-mediated endocytosis of asialoglycoproteins by rat hepatocytes: receptor-positive and receptor-negative endosomes. J Cell Biol. 1986 Mar;102(3):932–942. doi: 10.1083/jcb.102.3.932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mullock B. M., Branch W. J., van Schaik M., Gilbert L. K., Luzio J. P. Reconstitution of an endosome-lysosome interaction in a cell-free system. J Cell Biol. 1989 Jun;108(6):2093–2099. doi: 10.1083/jcb.108.6.2093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Murphy R. F., Powers S., Cantor C. R. Endosome pH measured in single cells by dual fluorescence flow cytometry: rapid acidification of insulin to pH 6. J Cell Biol. 1984 May;98(5):1757–1762. doi: 10.1083/jcb.98.5.1757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Opresko L. K., Karpf R. A. Specific proteolysis regulates fusion between endocytic compartments in Xenopus oocytes. Cell. 1987 Nov 20;51(4):557–568. doi: 10.1016/0092-8674(87)90125-5. [DOI] [PubMed] [Google Scholar]
  32. Roederer M., Bowser R., Murphy R. F. Kinetics and temperature dependence of exposure of endocytosed material to proteolytic enzymes and low pH: evidence for a maturation model for the formation of lysosomes. J Cell Physiol. 1987 May;131(2):200–209. doi: 10.1002/jcp.1041310209. [DOI] [PubMed] [Google Scholar]
  33. Salzman N. H., Maxfield F. R. Intracellular fusion of sequentially formed endocytic compartments. J Cell Biol. 1988 Apr;106(4):1083–1091. doi: 10.1083/jcb.106.4.1083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Samuelson A. C., Stockert R. J., Novikoff A. B., Novikoff P. M., Saez J. C., Spray D. C., Wolkoff A. W. Influence of cytosolic pH on receptor-mediated endocytosis of asialoorosomucoid. Am J Physiol. 1988 Jun;254(6 Pt 1):C829–C838. doi: 10.1152/ajpcell.1988.254.6.C829. [DOI] [PubMed] [Google Scholar]
  35. Sawatzki G., Anselstetter V., Kubanek B. Isolation of mouse transferrin using salting-out chromatography on Sepharose CL-6B. Biochim Biophys Acta. 1981 Jan 30;667(1):132–138. doi: 10.1016/0005-2795(81)90073-8. [DOI] [PubMed] [Google Scholar]
  36. Schmid S. L., Fuchs R., Male P., Mellman I. Two distinct subpopulations of endosomes involved in membrane recycling and transport to lysosomes. Cell. 1988 Jan 15;52(1):73–83. doi: 10.1016/0092-8674(88)90532-6. [DOI] [PubMed] [Google Scholar]
  37. Stahl P. D., Rodman J. S., Miller M. J., Schlesinger P. H. Evidence for receptor-mediated binding of glycoproteins, glycoconjugates, and lysosomal glycosidases by alveolar macrophages. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1399–1403. doi: 10.1073/pnas.75.3.1399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Stahl P., Schlesinger P. H., Sigardson E., Rodman J. S., Lee Y. C. Receptor-mediated pinocytosis of mannose glycoconjugates by macrophages: characterization and evidence for receptor recycling. Cell. 1980 Jan;19(1):207–215. doi: 10.1016/0092-8674(80)90402-x. [DOI] [PubMed] [Google Scholar]
  39. Stoorvogel W., Geuze H. J., Griffith J. M., Strous G. J. The pathways of endocytosed transferrin and secretory protein are connected in the trans-Golgi reticulum. J Cell Biol. 1988 Jun;106(6):1821–1829. doi: 10.1083/jcb.106.6.1821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Stoorvogel W., Geuze H. J., Strous G. J. Sorting of endocytosed transferrin and asialoglycoprotein occurs immediately after internalization in HepG2 cells. J Cell Biol. 1987 May;104(5):1261–1268. doi: 10.1083/jcb.104.5.1261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Stoscheck C. M., Carpenter G. Down regulation of epidermal growth factor receptors: direct demonstration of receptor degradation in human fibroblasts. J Cell Biol. 1984 Mar;98(3):1048–1053. doi: 10.1083/jcb.98.3.1048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Tycko B., Maxfield F. R. Rapid acidification of endocytic vesicles containing alpha 2-macroglobulin. Cell. 1982 Mar;28(3):643–651. doi: 10.1016/0092-8674(82)90219-7. [DOI] [PubMed] [Google Scholar]
  43. Vincent R., Nadeau D. A micromethod for the quantitation of cellular proteins in Percoll with the Coomassie brilliant blue dye-binding assay. Anal Biochem. 1983 Dec;135(2):355–362. doi: 10.1016/0003-2697(83)90696-6. [DOI] [PubMed] [Google Scholar]
  44. Ward D. M., Ajioka R., Kaplan J. Cohort movement of different ligands and receptors in the intracellular endocytic pathway of alveolar macrophages. J Biol Chem. 1989 May 15;264(14):8164–8170. [PubMed] [Google Scholar]
  45. Wiley H. S., Cunningham D. D. The endocytotic rate constant. A cellular parameter for quantitating receptor-mediated endocytosis. J Biol Chem. 1982 Apr 25;257(8):4222–4229. [PubMed] [Google Scholar]
  46. Wolkoff A. W., Klausner R. D., Ashwell G., Harford J. Intracellular segregation of asialoglycoproteins and their receptor: a prelysosomal event subsequent to dissociation of the ligand-receptor complex. J Cell Biol. 1984 Feb;98(2):375–381. doi: 10.1083/jcb.98.2.375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. van Renswoude J., Bridges K. R., Harford J. B., Klausner R. D. Receptor-mediated endocytosis of transferrin and the uptake of fe in K562 cells: identification of a nonlysosomal acidic compartment. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6186–6190. doi: 10.1073/pnas.79.20.6186. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES