Visualization of acidic organelles in intact cells by electron microscopy (original) (raw)

Abstract

We report the synthesis of a probe that permits the visualization by electron microscopy of acidic organelles in intact cells. This probe, 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP), is a basic congener of dinitrophenol that readily diffuses into intact cells. Its primary and tertiary amino groups (apparent pKa, 10.6) allow it to be concentrated in acidic organelles and to be retained there after fixation with aldehydes. The dinitroarene moiety of DAMP can then be localized with mouse monoclonal antibodies directed against dinitrophenol. The antibodies, in turn, can be visualized by light or electron microscopy by reaction with rabbit anti-mouse antibodies coupled to rhodamine or horseradish peroxidase, respectively. We have used these methods to show that DAMP concentrates in a variety of membrane-bound structures in cultured fibroblasts, including classic multivesicular bodies (resembling lysosomes), intermediate-sized vesicles with multiple shapes (resembling endosomes), and an abundant population of very small spherical vesicles. A small fraction of coated vesicles is labeled with DAMP. Labeling with DAMP does not occur when the pH gradient of fibroblasts is disrupted by the ionophore monensin or the weak base chloroquine. DAMP should be a useful probe for exploring the assembly, distribution, and function of acidic organelles by electron microscopy.

4838

Images in this article

Selected References

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

  1. ALLISON A. C., YOUNG M. R. UPTAKE OF DYES AND DRUGS BY LIVING CELLS IN CULTURE. Life Sci. 1964 Dec;3:1407–1414. doi: 10.1016/0024-3205(64)90082-7. [DOI] [PubMed] [Google Scholar]
  2. Anderson R. G., Brown M. S., Goldstein J. L. Inefficient internalization of receptor-bound low density lipoprotein in human carcinoma A-431 cells. J Cell Biol. 1981 Feb;88(2):441–452. doi: 10.1083/jcb.88.2.441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Basu S. K., Goldstein J. L., Anderson R. G., Brown M. S. Monensin interrupts the recycling of low density lipoprotein receptors in human fibroblasts. Cell. 1981 May;24(2):493–502. doi: 10.1016/0092-8674(81)90340-8. [DOI] [PubMed] [Google Scholar]
  5. Beisiegel U., Schneider W. J., Goldstein J. L., Anderson R. G., Brown M. S. Monoclonal antibodies to the low density lipoprotein receptor as probes for study of receptor-mediated endocytosis and the genetics of familial hypercholesterolemia. J Biol Chem. 1981 Nov 25;256(22):11923–11931. [PubMed] [Google Scholar]
  6. Forgac M., Cantley L., Wiedenmann B., Altstiel L., Branton D. Clathrin-coated vesicles contain an ATP-dependent proton pump. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1300–1303. doi: 10.1073/pnas.80.5.1300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Glickman J., Croen K., Kelly S., Al-Awqati Q. Golgi membranes contain an electrogenic H+ pump in parallel to a chloride conductance. J Cell Biol. 1983 Oct;97(4):1303–1308. doi: 10.1083/jcb.97.4.1303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Goldstein J. L., Basu S. K., Brown M. S. Receptor-mediated endocytosis of low-density lipoprotein in cultured cells. Methods Enzymol. 1983;98:241–260. doi: 10.1016/0076-6879(83)98152-1. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Louvard D., Reggio H., Warren G. Antibodies to the Golgi complex and the rough endoplasmic reticulum. J Cell Biol. 1982 Jan;92(1):92–107. doi: 10.1083/jcb.92.1.92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Marsh M., Bolzau E., Helenius A. Penetration of Semliki Forest virus from acidic prelysosomal vacuoles. Cell. 1983 Mar;32(3):931–940. doi: 10.1016/0092-8674(83)90078-8. [DOI] [PubMed] [Google Scholar]
  12. Ohkuma S., Poole B. Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3327–3331. doi: 10.1073/pnas.75.7.3327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Pressman B. C. Biological applications of ionophores. Annu Rev Biochem. 1976;45:501–530. doi: 10.1146/annurev.bi.45.070176.002441. [DOI] [PubMed] [Google Scholar]
  14. Scott M. G., Fleischman J. B. Preferential idiotype-isotype associations in antibodies to dinitrophenyl antigens. J Immunol. 1982 Jun;128(6):2622–2628. [PubMed] [Google Scholar]
  15. Stone D. K., Xie X. S., Racker E. An ATP-driven proton pump in clathrin-coated vesicles. J Biol Chem. 1983 Apr 10;258(7):4059–4062. [PubMed] [Google Scholar]
  16. 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]
  17. de Duve C., de Barsy T., Poole B., Trouet A., Tulkens P., Van Hoof F. Commentary. Lysosomotropic agents. Biochem Pharmacol. 1974 Sep 15;23(18):2495–2531. doi: 10.1016/0006-2952(74)90174-9. [DOI] [PubMed] [Google Scholar]