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. 1995 Jun 1;129(5):1229–1240. doi: 10.1083/jcb.129.5.1229

"Synchronized" endocytosis and intracellular sorting in alveolar macrophages: the early sorting endosome is a transient organelle

PMCID: PMC2120465  PMID: 7775570

Abstract

Incubation of alveolar macrophages in hypoosmotic K(+)-containing buffers results in persistent cell swelling and an inability to undergo regulatory volume decrease. We demonstrate that cells incubated in hypo- K+ show an inhibition of endocytosis without any observed alteration in recycling. The inhibition of endocytosis affected all forms of membrane internalization, receptor and fluid phase. Both increased cell volume and the inhibition of endocytosis could be released upon return of cells to iso-Na+ buffers. The ability to synchronize the endocytic apparatus allowed us to examine hypotheses regarding the origin and maturation of endocytic vesicles. Incubation in hypo-K+ buffers had no effect on the delivery of ligands to degradative compartments or on the return of previously internalized receptors to the cell surface. Thus, membrane recycling and movement of internalized components to lysosomes occurred in the absence of continued membrane influx. We also demonstrate that fluorescent lipids, that had been incorporated into early endosomes, returned to the cell surface upon exposure of cells to hypo-K+ buffers. These results indicate that the early sorting endosome is a transient structure, whose existence depends upon continued membrane internalization. Our data supports the hypothesis that the transfer of material to lysosomes can best be explained by the continuous maturation of endosomes.

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Selected References

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  1. Aniento F., Emans N., Griffiths G., Gruenberg J. Cytoplasmic dynein-dependent vesicular transport from early to late endosomes. J Cell Biol. 1993 Dec;123(6 Pt 1):1373–1387. doi: 10.1083/jcb.123.6.1373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Bowser R., Murphy R. F. Kinetics of hydrolysis of endocytosed substrates by mammalian cultured cells: early introduction of lysosomal enzymes into the endocytic pathway. J Cell Physiol. 1990 Apr;143(1):110–117. doi: 10.1002/jcp.1041430115. [DOI] [PubMed] [Google Scholar]
  4. Braun M., Waheed A., von Figura K. Lysosomal acid phosphatase is transported to lysosomes via the cell surface. EMBO J. 1989 Dec 1;8(12):3633–3640. doi: 10.1002/j.1460-2075.1989.tb08537.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Buys S. S., Novak J. M., Gren L. H., Kaplan J. Effect of volume and pH on surface receptor number in macrophages. J Cell Physiol. 1989 Aug;140(2):371–378. doi: 10.1002/jcp.1041400223. [DOI] [PubMed] [Google Scholar]
  6. Casciola-Rosen L. A., Hubbard A. L. Lumenal labeling of rat hepatocyte early endosomes. Presence of multiple membrane receptors and the Na+,K(+)-ATPase. J Biol Chem. 1992 Apr 25;267(12):8213–8221. [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. 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]
  10. Dunn K. W., Maxfield F. R. Delivery of ligands from sorting endosomes to late endosomes occurs by maturation of sorting endosomes. J Cell Biol. 1992 Apr;117(2):301–310. doi: 10.1083/jcb.117.2.301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Felder S., Miller K., Moehren G., Ullrich A., Schlessinger J., Hopkins C. R. Kinase activity controls the sorting of the epidermal growth factor receptor within the multivesicular body. Cell. 1990 May 18;61(4):623–634. doi: 10.1016/0092-8674(90)90474-s. [DOI] [PubMed] [Google Scholar]
  12. French A. R., Sudlow G. P., Wiley H. S., Lauffenburger D. A. Postendocytic trafficking of epidermal growth factor-receptor complexes is mediated through saturable and specific endosomal interactions. J Biol Chem. 1994 Jun 3;269(22):15749–15755. [PubMed] [Google Scholar]
  13. Geuze H. J., Slot J. W., Strous G. J., Lodish H. F., Schwartz A. L. Intracellular site of asialoglycoprotein receptor-ligand uncoupling: double-label immunoelectron microscopy during receptor-mediated endocytosis. Cell. 1983 Jan;32(1):277–287. doi: 10.1016/0092-8674(83)90518-4. [DOI] [PubMed] [Google Scholar]
  14. Griffiths G., Gruenberg J. The arguments for pre-existing early and late endosomes. Trends Cell Biol. 1991 Jul;1(1):5–9. doi: 10.1016/0962-8924(91)90047-d. [DOI] [PubMed] [Google Scholar]
  15. Grinstein S., Dupre A., Rothstein A. Volume regulation by human lymphocytes. Role of calcium. J Gen Physiol. 1982 May;79(5):849–868. doi: 10.1085/jgp.79.5.849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Guagliardi L. E., Koppelman B., Blum J. S., Marks M. S., Cresswell P., Brodsky F. M. Co-localization of molecules involved in antigen processing and presentation in an early endocytic compartment. Nature. 1990 Jan 11;343(6254):133–139. doi: 10.1038/343133a0. [DOI] [PubMed] [Google Scholar]
  17. Herbst J. J., Opresko L. K., Walsh B. J., Lauffenburger D. A., Wiley H. S. Regulation of postendocytic trafficking of the epidermal growth factor receptor through endosomal retention. J Biol Chem. 1994 Apr 29;269(17):12865–12873. [PubMed] [Google Scholar]
  18. Hopkins C. R., Gibson A., Shipman M., Miller K. Movement of internalized ligand-receptor complexes along a continuous endosomal reticulum. Nature. 1990 Jul 26;346(6282):335–339. doi: 10.1038/346335a0. [DOI] [PubMed] [Google Scholar]
  19. Hopkins C. R., Gibson A., Shipman M., Strickland D. K., Trowbridge I. S. In migrating fibroblasts, recycling receptors are concentrated in narrow tubules in the pericentriolar area, and then routed to the plasma membrane of the leading lamella. J Cell Biol. 1994 Jun;125(6):1265–1274. doi: 10.1083/jcb.125.6.1265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jing S. Q., Spencer T., Miller K., Hopkins C., Trowbridge I. S. Role of the human transferrin receptor cytoplasmic domain in endocytosis: localization of a specific signal sequence for internalization. J Cell Biol. 1990 Feb;110(2):283–294. doi: 10.1083/jcb.110.2.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. 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]
  24. Kessell I., Holst B. D., Roth T. F. Membranous intermediates in endocytosis are labile, as shown in a temperature-sensitive mutant. Proc Natl Acad Sci U S A. 1989 Jul;86(13):4968–4972. doi: 10.1073/pnas.86.13.4968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Klausner R. D., Donaldson J. G., Lippincott-Schwartz J. Brefeldin A: insights into the control of membrane traffic and organelle structure. J Cell Biol. 1992 Mar;116(5):1071–1080. doi: 10.1083/jcb.116.5.1071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Koval M., Pagano R. E. Lipid recycling between the plasma membrane and intracellular compartments: transport and metabolism of fluorescent sphingomyelin analogues in cultured fibroblasts. J Cell Biol. 1989 Jun;108(6):2169–2181. doi: 10.1083/jcb.108.6.2169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Martin O. C., Pagano R. E. Internalization and sorting of a fluorescent analogue of glucosylceramide to the Golgi apparatus of human skin fibroblasts: utilization of endocytic and nonendocytic transport mechanisms. J Cell Biol. 1994 May;125(4):769–781. doi: 10.1083/jcb.125.4.769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mayor S., Presley J. F., Maxfield F. R. Sorting of membrane components from endosomes and subsequent recycling to the cell surface occurs by a bulk flow process. J Cell Biol. 1993 Jun;121(6):1257–1269. doi: 10.1083/jcb.121.6.1257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. McGraw T. E., Maxfield F. R. Human transferrin receptor internalization is partially dependent upon an aromatic amino acid on the cytoplasmic domain. Cell Regul. 1990 Mar;1(4):369–377. doi: 10.1091/mbc.1.4.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Murphy R. F. Maturation models for endosome and lysosome biogenesis. Trends Cell Biol. 1991 Oct;1(4):77–82. doi: 10.1016/0962-8924(91)90022-2. [DOI] [PubMed] [Google Scholar]
  32. Naim H. Y., Roth M. G. Characteristics of the internalization signal in the Y543 influenza virus hemagglutinin suggest a model for recognition of internalization signals containing tyrosine. J Biol Chem. 1994 Feb 11;269(6):3928–3933. [PubMed] [Google Scholar]
  33. Novak J. M., Cala P. M., Ward D. M., Buys S. S., Kaplan J. Regulatory volume decrease in alveolar macrophages: cation loss is not correlated with changes in membrane recycling. J Cell Physiol. 1988 Nov;137(2):243–250. doi: 10.1002/jcp.1041370206. [DOI] [PubMed] [Google Scholar]
  34. Novak J. M., Ward D. M., Buys S. S., Kaplan J. Effect of hypo-osmotic incubation on membrane recycling. J Cell Physiol. 1988 Nov;137(2):235–242. doi: 10.1002/jcp.1041370205. [DOI] [PubMed] [Google Scholar]
  35. Opresko L. K., Wiley H. S. Receptor-mediated endocytosis in Xenopus oocytes. II. Evidence for two novel mechanisms of hormonal regulation. J Biol Chem. 1987 Mar 25;262(9):4116–4123. [PubMed] [Google Scholar]
  36. Pagano R. E., Martin O. C., Kang H. C., Haugland R. P. A novel fluorescent ceramide analogue for studying membrane traffic in animal cells: accumulation at the Golgi apparatus results in altered spectral properties of the sphingolipid precursor. J Cell Biol. 1991 Jun;113(6):1267–1279. doi: 10.1083/jcb.113.6.1267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Roederer M., Barry J. R., Wilson R. B., Murphy R. F. Endosomes can undergo an ATP-dependent density increase in the absence of dense lysosomes. Eur J Cell Biol. 1990 Apr;51(2):229–234. [PubMed] [Google Scholar]
  38. Rothman J. E., Orci L. Molecular dissection of the secretory pathway. Nature. 1992 Jan 30;355(6359):409–415. doi: 10.1038/355409a0. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. Stoorvogel W., Strous G. J., Geuze H. J., Oorschot V., Schwartz A. L. Late endosomes derive from early endosomes by maturation. Cell. 1991 May 3;65(3):417–427. doi: 10.1016/0092-8674(91)90459-c. [DOI] [PubMed] [Google Scholar]
  42. Sukhorukov V. L., Arnold W. M., Zimmermann U. Hypotonically induced changes in the plasma membrane of cultured mammalian cells. J Membr Biol. 1993 Feb;132(1):27–40. doi: 10.1007/BF00233049. [DOI] [PubMed] [Google Scholar]
  43. Tooze J., Hollinshead M. Tubular early endosomal networks in AtT20 and other cells. J Cell Biol. 1991 Nov;115(3):635–653. doi: 10.1083/jcb.115.3.635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Tsuruhara T., Koenig J. H., Ikeda K. Synchronized endocytosis studied in the oocyte of a temperature-sensitive mutant of Drosophila melanogaster. Cell Tissue Res. 1990 Feb;259(2):199–207. doi: 10.1007/BF00318441. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Ward D. M., Hackenyos D. P., Davis-Kaplan S., Kaplan J. Inhibition of late endosome-lysosome fusion: studies on the mechanism by which isotonic-K+ buffers alter intracellular ligand movement. J Cell Physiol. 1990 Dec;145(3):522–530. doi: 10.1002/jcp.1041450319. [DOI] [PubMed] [Google Scholar]
  47. Ward D. M., Hackenyos D. P., Kaplan J. Fusion of sequentially internalized vesicles in alveolar macrophages. J Cell Biol. 1990 Apr;110(4):1013–1022. doi: 10.1083/jcb.110.4.1013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Ward D. M., Kaplan J. The rate of internalization of different receptor-ligand complexes in alveolar macrophages is receptor-specific. Biochem J. 1990 Sep 1;270(2):369–374. doi: 10.1042/bj2700369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Ward J. H., Kushner J. P., Kaplan J. Regulation of HeLa cell transferrin receptors. J Biol Chem. 1982 Sep 10;257(17):10317–10323. [PubMed] [Google Scholar]
  50. Wiley H. S., Herbst J. J., Walsh B. J., Lauffenburger D. A., Rosenfeld M. G., Gill G. N. The role of tyrosine kinase activity in endocytosis, compartmentation, and down-regulation of the epidermal growth factor receptor. J Biol Chem. 1991 Jun 15;266(17):11083–11094. [PubMed] [Google Scholar]
  51. van Deurs B., Petersen O. W., Olsnes S., Sandvig K. The ways of endocytosis. Int Rev Cytol. 1989;117:131–177. doi: 10.1016/s0074-7696(08)61336-4. [DOI] [PubMed] [Google Scholar]

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