Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1986 May 1;102(5):1797–1812. doi: 10.1083/jcb.102.5.1797

Redistribution and shedding of flagellar membrane glycoproteins visualized using an anti-carbohydrate monoclonal antibody and concanavalin A

PMCID: PMC2114210  PMID: 3009491

Abstract

Two carbohydrate-binding probes, the lectin concanavalin A and an anti- carbohydrate monoclonal antibody designated FMG-1, have been used to study the distribution of their respective epitopes on the surface of Chlamydomonas reinhardtii, strain pf-18. Both of these ligands bind uniformly to the external surface of the flagellar membrane and the general cell body plasma membrane, although the labeling is more intense on the flagellar membrane. In addition, both ligands cross- react with cell wall glycoproteins. With respect to the flagellar membrane, both concanavalin A and the FMG-1 monoclonal antibody bind preferentially to the principal high molecular weight glycoproteins migrating with an apparent molecular weight of 350,000 although there is, in addition, cross-reactivity with a number of minor glycoproteins. Western blots of V-8 protease digests of the high molecular weight flagellar glycoproteins indicate that the epitopes recognized by the lectin and the antibody are both repeated multiple times within the glycoproteins and occur together, although the lectin and the antibody do not compete for the same binding sites. Incubation of live cells with the monoclonal antibody or lectin at 4 degrees C results in a uniform labeling of the flagellar surface; upon warming of the cells, these ligands are redistributed along the flagellar surface in a characteristic manner. All of the flagellar surface-bound antibody or lectin collects into a single aggregate at the tip of each flagellum; this aggregate subsequently migrates to the base of the flagellum, where it is shed into the medium. The rate of redistribution is temperature dependent and the glycoproteins recognized by these ligands co-redistribute with the lectin or monoclonal antibody. This dynamic flagellar surface phenomenon bears a striking resemblance to the capping phenomenon that has been described in numerous mammalian cell types. However, it occurs on a structure (the flagellum) that lacks most of the cytoskeletal components generally associated with capping in other systems. The FMG-1 monoclonal antibody inhibits flagellar surface motility visualized as the rapid, bidirectional translocation of polystyrene microspheres.

Full Text

The Full Text of this article is available as a PDF (3.9 MB).

Selected References

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

  1. Adair W. S., Hwang C., Goodenough U. W. Identification and visualization of the sexual agglutinin from the mating-type plus flagellar membrane of Chlamydomonas. Cell. 1983 May;33(1):183–193. doi: 10.1016/0092-8674(83)90347-1. [DOI] [PubMed] [Google Scholar]
  2. Adams G. M., Huang B., Piperno G., Luck D. J. Central-pair microtubular complex of Chlamydomonas flagella: polypeptide composition as revealed by analysis of mutants. J Cell Biol. 1981 Oct;91(1):69–76. doi: 10.1083/jcb.91.1.69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Adoutte A., Ramanathan R., Lewis R. M., Dute R. R., Ling K. Y., Kung C., Nelson D. L. Biochemical studies of the excitable membrane of Paramecium tetraurelia. III. Proteins of cilia and ciliary membranes. J Cell Biol. 1980 Mar;84(3):717–738. doi: 10.1083/jcb.84.3.717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bergman K., Goodenough U. W., Goodenough D. A., Jawitz J., Martin H. Gametic differentiation in Chlamydomonas reinhardtii. II. Flagellar membranes and the agglutination reaction. J Cell Biol. 1975 Dec;67(3):606–622. doi: 10.1083/jcb.67.3.606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bloodgood R. A. Dynamic properties of the flagellar surface. Symp Soc Exp Biol. 1982;35:353–380. [PubMed] [Google Scholar]
  6. Bloodgood R. A., Leffler E. M., Bojczuk A. T. Reversible inhibition of Chlamydomonas flagellar surface motility. J Cell Biol. 1979 Sep;82(3):664–674. doi: 10.1083/jcb.82.3.664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bloodgood R. A., May G. S. Functional modification of the Chlamydomonas flagellar surface. J Cell Biol. 1982 Apr;93(1):88–96. doi: 10.1083/jcb.93.1.88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bloodgood R. A. Motility occurring in association with the surface of the Chlamydomonas flagellum. J Cell Biol. 1977 Dec;75(3):983–989. doi: 10.1083/jcb.75.3.983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bloodgood R. A. Preferential turnover of membrane proteins in the intact Chlamydomonas flagellum. Exp Cell Res. 1984 Feb;150(2):488–493. doi: 10.1016/0014-4827(84)90594-9. [DOI] [PubMed] [Google Scholar]
  10. Bloodgood R. A., Workman L. J. A flagellar surface glycoprotein mediating cell-substrate interaction in Chlamydomonas. Cell Motil. 1984;4(2):77–87. doi: 10.1002/cm.970040202. [DOI] [PubMed] [Google Scholar]
  11. Bourguignon L. Y., Bourguignon G. J. Capping and the cytoskeleton. Int Rev Cytol. 1984;87:195–224. doi: 10.1016/s0074-7696(08)62443-2. [DOI] [PubMed] [Google Scholar]
  12. Bruck C., Portetelle D., Glineur C., Bollen A. One-step purification of mouse monoclonal antibodies from ascitic fluid by DEAE Affi-Gel blue chromatography. J Immunol Methods. 1982 Sep 30;53(3):313–319. doi: 10.1016/0022-1759(82)90178-8. [DOI] [PubMed] [Google Scholar]
  13. Claes H. Non-specific stimulation of the autolytic system in gametes from Chlamydomonas reinhardii. Exp Cell Res. 1977 Aug;108(1):221–229. [PubMed] [Google Scholar]
  14. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
  15. Dentler W. L. Microtubule-membrane interactions in cilia and flagella. Int Rev Cytol. 1981;72:1–47. doi: 10.1016/s0074-7696(08)61193-6. [DOI] [PubMed] [Google Scholar]
  16. Ey P. L., Prowse S. J., Jenkin C. R. Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-sepharose. Immunochemistry. 1978 Jul;15(7):429–436. doi: 10.1016/0161-5890(78)90070-6. [DOI] [PubMed] [Google Scholar]
  17. Forest C. L., Goodenough D. A., Goodenough U. W. Flagellar membrane agglutination and sexual signaling in the conditional GAM-1 mutant of Chlamydomonas. J Cell Biol. 1978 Oct;79(1):74–84. doi: 10.1083/jcb.79.1.74. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Goodenough U. W., Adair W. S., Caligor E., Forest C. L., Hoffman J. L., Mesland D. A., Spath S. Membrane-membrane and membrane-ligand interactions in Chlamydomonas mating. Soc Gen Physiol Ser. 1980;34:131–152. [PubMed] [Google Scholar]
  19. Goodenough U. W., Jurivich D. Tipping and mating-structure activation induced in Chlamydomonas gametes by flagellar membrane antisera. J Cell Biol. 1978 Dec;79(3):680–693. doi: 10.1083/jcb.79.3.680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hoffman J. L., Goodenough U. W. Experimental dissection of flagellar surface motility in Chlamydomonas. J Cell Biol. 1980 Aug;86(2):656–665. doi: 10.1083/jcb.86.2.656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jarvik J. W., Rosenbaum J. L. Oversized flagellar membrane protein in paralyzed mutants of Chlamydomonas reinhardrii. J Cell Biol. 1980 May;85(2):258–272. doi: 10.1083/jcb.85.2.258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. McLEAN R. J., Laurendi C. J., Brown R. M., Jr The relationship of gamone to the mating reaction in Chlamydomonas moewusii. Proc Natl Acad Sci U S A. 1974 Jul;71(7):2610–2613. doi: 10.1073/pnas.71.7.2610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McLean R. J., Brown R. M. Cell surface differentiation of Chlamydomonas during gametogenesis. I. Mating and concanavalin A agglutinability. Dev Biol. 1974 Feb;36(2):279–285. doi: 10.1016/0012-1606(74)90051-7. [DOI] [PubMed] [Google Scholar]
  25. Oakley B. R., Kirsch D. R., Morris N. R. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem. 1980 Jul 1;105(2):361–363. doi: 10.1016/0003-2697(80)90470-4. [DOI] [PubMed] [Google Scholar]
  26. Olden K., Parent J. B., White S. L. Carbohydrate moieties of glycoproteins. A re-evaluation of their function. Biochim Biophys Acta. 1982 May 12;650(4):209–232. doi: 10.1016/0304-4157(82)90017-x. [DOI] [PubMed] [Google Scholar]
  27. Parham P. On the fragmentation of monoclonal IgG1, IgG2a, and IgG2b from BALB/c mice. J Immunol. 1983 Dec;131(6):2895–2902. [PubMed] [Google Scholar]
  28. Piperno G., Luck D. J. An actin-like protein is a component of axonemes from Chlamydomonas flagella. J Biol Chem. 1979 Apr 10;254(7):2187–2190. [PubMed] [Google Scholar]
  29. Ray D. A., Gibor A. Tunicamycin-sensitive glycoproteins involved in the mating of Chlamydomonas reinhardi. Exp Cell Res. 1982 Oct;141(2):245–252. doi: 10.1016/0014-4827(82)90212-9. [DOI] [PubMed] [Google Scholar]
  30. Remillard S. P., Witman G. B. Synthesis, transport, and utilization of specific flagellar proteins during flagellar regeneration in Chlamydomonas. J Cell Biol. 1982 Jun;93(3):615–631. doi: 10.1083/jcb.93.3.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ringo D. L. Flagellar motion and fine structure of the flagellar apparatus in Chlamydomonas. J Cell Biol. 1967 Jun;33(3):543–571. doi: 10.1083/jcb.33.3.543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. SAGER R., GRANICK S. Nutritional control of sexuality in Chlamydomonas reinhardi. J Gen Physiol. 1954 Jul 20;37(6):729–742. doi: 10.1085/jgp.37.6.729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. SAGER R., GRANICK S. Nutritional studies with Chlamydomonas reinhardi. Ann N Y Acad Sci. 1953 Oct 14;56(5):831–838. doi: 10.1111/j.1749-6632.1953.tb30261.x. [DOI] [PubMed] [Google Scholar]
  34. Salisbury J. L., Condeelis J. S., Satir P. Role of coated vesicles, microfilaments, and calmodulin in receptor-mediated endocytosis by cultured B lymphoblastoid cells. J Cell Biol. 1980 Oct;87(1):132–141. doi: 10.1083/jcb.87.1.132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sharon N., Lis H. Glycoproteins: research booming on long-ignored ubiquitous compounds. Mol Cell Biochem. 1982 Feb 19;42(3):167–187. doi: 10.1007/BF00238511. [DOI] [PubMed] [Google Scholar]
  36. Snell W. J. Mating in Chlamydomonas: a system for the study of specific cell adhesion. I. Ultrastructural and electrophoretic analyses of flagellar surface components involved in adhesion. J Cell Biol. 1976 Jan;68(1):48–69. doi: 10.1083/jcb.68.1.48. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Snell W. K. Flagellar adhesion and deadhesion in Chlamydomonas gametes: effects of tunicamycin and observations on flagellar tip morphology. J Supramol Struct Cell Biochem. 1981;16(4):371–376. doi: 10.1002/jsscb.1981.380160407. [DOI] [PubMed] [Google Scholar]
  38. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Witman G. B., Carlson K., Berliner J., Rosenbaum J. L. Chlamydomonas flagella. I. Isolation and electrophoretic analysis of microtubules, matrix, membranes, and mastigonemes. J Cell Biol. 1972 Sep;54(3):507–539. doi: 10.1083/jcb.54.3.507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Woodward M. P., Young W. W., Jr, Bloodgood R. A. Detection of monoclonal antibodies specific for carbohydrate epitopes using periodate oxidation. J Immunol Methods. 1985 Apr 8;78(1):143–153. doi: 10.1016/0022-1759(85)90337-0. [DOI] [PubMed] [Google Scholar]
  41. van den Ende H. Sexual agglutination in chlamydomonads. Adv Microb Physiol. 1985;26:89–123. doi: 10.1016/s0065-2911(08)60395-1. [DOI] [PubMed] [Google Scholar]

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

RESOURCES