Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1981 Apr;1(4):358–369. doi: 10.1128/mcb.1.4.358

Localization of lipophosphonoglycan in membranes of Acanthamoeba by using specific antibodies.

C F Bailey 1, B Bowers 1
PMCID: PMC369684  PMID: 6765602

Abstract

Antibodies against two electrophoretically distinct forms of lipophosphonoglycan (LPG) were produced in rabbits. Antibody specificity was demonstrated by the coupled antibody 125I-protein A assay (Adair et al., J. Cell Biol. 79:281-285, 1978). Indirect immunofluorescent labeling of intact Acanthamoeba showed that antibodies to both LPG components had the same uniform distribution on the cell surface. Both antibodies also bound to the cytoplasmic surface of isolated phagosomes. The location of LPG in other membranes of the amoeba was demonstrated on sections by the unlabeled antibody method. Although LPG was absent from the nuclear membrane, virtually all of the internal vacuole membranes were labeled, including the contractile vacuole. Antibodies directed against LPG were utilized to label lipophosphonoglycan in the plasma membrane of living amoebae. Labeled membrane was internalized and then localized by immunofluorescence in cytoplasmic vacuoles within 10 min of incubation. Although these results are evidence for exchange between plasma and cytoplasmic vacuolar membranes, the contractile vacuole remained unlabeled and can be considered, therefore, a separate membrane compartment. Concanavalin A also was bound and internalized by the amoeba, but electron microscopy showed that this label caused pronounced membrane perturbation, limiting its usefulness as a membrane marker in this system.

Full text

PDF
358

Images in this article

360Image
on p.360
361Image
on p.361
361Image
on p.361
362Image
on p.362
363Image
on p.363
364Image
on p.364
365Image
on p.365
366Image
on p.366
367Image
on p.367

Selected References

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

  1. Adair W. S., Jurivich D., Goodenough U. W. Localization of cellular antigens in sodium dodecyl sulfate-polyacrylamide gels. J Cell Biol. 1978 Oct;79(1):281–285. doi: 10.1083/jcb.79.1.281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bowers B. A morphological study of plasma and phagosome membranes during endocytosis in Acanthamoeba. J Cell Biol. 1980 Feb;84(2):246–260. doi: 10.1083/jcb.84.2.246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bowers B., Korn E. D. Localization of lipophosphonoglycan on both sides of Acanthamoeba plasma membrane. J Cell Biol. 1974 Aug;62(2):533–540. doi: 10.1083/jcb.62.2.533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bowers B., Olszewski T. E., Hyde J. Morphometric analysis of volumes and surface areas in membrane compartments during endocytosis in Acanthamoeba. J Cell Biol. 1981 Mar;88(3):509–515. doi: 10.1083/jcb.88.3.509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bowers B., Olszewski T. E. Pinocytosis in Acanthamoeba castellanii. Kinetics and morphology. J Cell Biol. 1972 Jun;53(3):681–694. doi: 10.1083/jcb.53.3.681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brown S. S., Revel J. P. Reversibility of cell surface label rearrangement. J Cell Biol. 1976 Mar;68(3):629–641. doi: 10.1083/jcb.68.3.629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dearborn D. G., Smith S., Korn E. D. Lipophosphonoglycan of the plasma membrane of A canthamoeba castellanii. Inositol and phytosphingosine content and general structural features. J Biol Chem. 1976 May 25;251(10):2976–2982. [PubMed] [Google Scholar]
  8. Flickinger C. J. The relation between the Golgi apparatus, cell surface, and cytoplasmic vesicles in amoebae studied by electron microscope radioautography. Exp Cell Res. 1975 Nov;96(1):189–201. doi: 10.1016/s0014-4827(75)80051-6. [DOI] [PubMed] [Google Scholar]
  9. GREENWOOD F. C., HUNTER W. M., GLOVER J. S. THE PREPARATION OF I-131-LABELLED HUMAN GROWTH HORMONE OF HIGH SPECIFIC RADIOACTIVITY. Biochem J. 1963 Oct;89:114–123. doi: 10.1042/bj0890114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Geoghegan W. D., Ackerman G. A. Adsorption of horseradish peroxidase, ovomucoid and anti-immunoglobulin to colloidal gold for the indirect detection of concanavalin A, wheat germ agglutinin and goat anti-human immunoglobulin G on cell surfaces at the electron microscopic level: a new method, theory and application. J Histochem Cytochem. 1977 Nov;25(11):1187–1200. doi: 10.1177/25.11.21217. [DOI] [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. Hellio R., Ryter A. Relationships between anionic sites and lectin receptors in the plasma membrane of Dictyostelium discoideum and their role in phagocytosis. J Cell Sci. 1980 Feb;41:89–104. doi: 10.1242/jcs.41.1.89. [DOI] [PubMed] [Google Scholar]
  13. Inoue K., Graf L., Rapport M. M. Immunochemical studies of organ and tumor lipids. XIX. Cytolytic action of antibodies directed against cytolipin R. J Lipid Res. 1972 Jan;13(1):119–127. [PubMed] [Google Scholar]
  14. Inoue K., Nojima S. Immunochemical studies of phospholipids. 3. Production of antibody to cardiolipin. Biochim Biophys Acta. 1967 Oct 2;144(2):409–414. [PubMed] [Google Scholar]
  15. Karnovsky M. J., Unanue E. R. Mapping and migration of lymphocyte surface macromolecules. Fed Proc. 1973 Jan;32(1):55–59. [PubMed] [Google Scholar]
  16. King C. A., Preston T. M. Studies of anionic sites on the cell surface of the amoeba Naegleria gruberi using cationized ferritin. J Cell Sci. 1977 Dec;28:133–149. doi: 10.1242/jcs.28.1.133. [DOI] [PubMed] [Google Scholar]
  17. Korn E. D., Dearborn D. G., Wright P. L. Lipophosphonoglycan of the plasma membrance of Acanthamoeba castellanii. Isolation from whole amoebae and identification of the water-soluble products of acid hydrolysis. J Biol Chem. 1974 Jun 10;249(11):3335–3341. [PubMed] [Google Scholar]
  18. Korn E. D. The isolation of the amoeba plasma membrane and the use of latex beads for the isolation of phagocytic vacuole (phagosome) membranes from amoebae including the culture techniques for amoebae. Methods Enzymol. 1974;31:686–698. doi: 10.1016/0076-6879(74)31074-9. [DOI] [PubMed] [Google Scholar]
  19. Korn E. D., Wright P. L. Macromolecular composition of an amoeba plasma membrane. J Biol Chem. 1973 Jan 25;248(2):439–447. [PubMed] [Google Scholar]
  20. LUFT J. H. Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol. 1961 Feb;9:409–414. doi: 10.1083/jcb.9.2.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  22. Rapport M. M., Graf L. Immunochemical reactions of lipids. Prog Allergy. 1969;13:273–331. [PubMed] [Google Scholar]
  23. Roth J., Bendayan M., Orci L. Ultrastructural localization of intracellular antigens by the use of protein A-gold complex. J Histochem Cytochem. 1978 Dec;26(12):1074–1081. doi: 10.1177/26.12.366014. [DOI] [PubMed] [Google Scholar]
  24. Ryter A., Hellio R. Electron-microscope study of Dictyostelium discoideum plasma membrane and its modifications during and after phagocytosis. J Cell Sci. 1980 Feb;41:75–88. doi: 10.1242/jcs.41.1.75. [DOI] [PubMed] [Google Scholar]
  25. Schachter H., Jabbal I., Hudgin R. L., Pinteric L., McGuire E. J., Roseman S. Intracellular localization of liver sugar nucleotide glycoprotein glycosyltransferases in a Golgi-rich fraction. J Biol Chem. 1970 Mar 10;245(5):1090–1100. [PubMed] [Google Scholar]
  26. Schlessinger J., Shechter Y., Willingham M. C., Pastan I. Direct visualization of binding, aggregation, and internalization of insulin and epidermal growth factor on living fibroblastic cells. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2659–2663. doi: 10.1073/pnas.75.6.2659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Silva P. P., Martínez-Palomo A., Gonzalez-Robles A. Membrane structure and surface coat of Entamoeba histolytica. Topochemistry and dynamics of the cell surface: cap formation and microexudate. J Cell Biol. 1975 Mar;64(3):538–550. doi: 10.1083/jcb.64.3.538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Stackpole C. W., Jacobson J. B., Lardis M. P. Two distinct types of capping of surface receptors on mouse lymphoid cells. Nature. 1974 Mar 15;248(445):232–234. doi: 10.1038/248232a0. [DOI] [PubMed] [Google Scholar]
  29. Ulsamer A. G., Wright P. L., Wetzel M. G., Korn E. D. Plasma and phagosome membranes of Acanthamoeba castellanii. J Cell Biol. 1971 Oct;51(1):193–215. doi: 10.1083/jcb.51.1.193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Verkleij A. J., Momvers C., Leunissen-Bijvelt J., Ververgaert P. H. Lipidic intramembranous particles. Nature. 1979 May 10;279(5709):162–163. doi: 10.1038/279162a0. [DOI] [PubMed] [Google Scholar]
  31. West C. M., McMahon D., Molday R. S. Identification of glycoproteins, using lectins as probes, in plasma membranes from Dictyostelium discoideum and human erythrocytes. J Biol Chem. 1978 Mar 10;253(5):1716–1724. [PubMed] [Google Scholar]
  32. Wetzel M. G., Korn E. D. Phagocytosis of latex beads by Acahamoeba castellanii (Neff). 3. Isolation of the phagocytic vesicles and their membranes. J Cell Biol. 1969 Oct;43(1):90–104. doi: 10.1083/jcb.43.1.90. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES