Hostname: page-component-78c5997874-lj6df Total loading time: 0 Render date: 2024-11-03T00:21:52.819Z Has data issue: false hasContentIssue false
  • English
  • Français

Immunological characterization of cytoskeletal proteins associated with the basal body, axoneme and flagellum attachment zone of Trypanosoma brucei

Published online by Cambridge University Press:  06 April 2009

R. Woodward ,
M. J. Carden  and
K. Gull
R. Woodward
Affiliation:
Research School of Biosciences, Biological Laboratory, University of Kent at Canterbury, Canterbury CT2 7NJ
M. J. Carden
Affiliation:
Research School of Biosciences, Biological Laboratory, University of Kent at Canterbury, Canterbury CT2 7NJ
K. Gull
Affiliation:
School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester M13 9PT
Get access Rights & Permissions [Opens in a new window]

Summary

The monoclonal antibody BS7, raised to bovine sperm flagellum cytoskeletal antigens in a previous study, is here reported to detect flagellum-associated structures in Trypanosoma brucei and Crithidia fasciculata. Immunoblotting showed that BS7 cross-reacts with several cytoskeletal T. brucei proteins but phosphatase treatment did not diminish this complex immunoblot reactivity. To characterize further the cross-reactive proteins recognized in T. brucei-cytoskeletons by BS7 each was excised from preparative gels and used as an immunogen for antiserum production. Two proteins, with apparent sizes around 43 and 47 kDa, produced antisera shown to be monospecific by immunoblotting total T. brucei flagellum preparations. Each of these detected the basal body-associated immunofluorescence in T. brucei. Identification of the smaller, 43 kDa, component as a basal body-associated product was supported by the behaviour of a second monoclonal antibody, BBA4, which was also shown to detect the T. brucei basal body complex by immunofluorescence and immunoblots the 43 kDa polypeptide. These observations reveal new components of the trypanosome cytoskeleton. Also, they provide a further example of an immunological approach for identification of interesting, rare components of the T. brucei cytoskeleton starting from a complex mixture of proteins.

Keywords

trypanosomecytoskeletonbasal bodiesflagellumantibody.
Type
Research Article
Information
Parasitology , Volume 111 , Issue 1 , July 1995 , pp. 77 - 85
Copyright
Copyright © Cambridge University Press 1995

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Birkett, C. R., Parma, A. E., Gerke-Bonet, R., Woodward, R. & Gull, K. (1992). Isolation of cDNA clones encoding proteins of complex structures: analysis of the Trypanosoma brucei cytoskeleton. Gene 110, 65–70.CrossRefGoogle ScholarPubMed
Brun, R. & Schonenberger, M. (1979). Cultivation and in vitro cloning of procyclic culture forms of Trypanosoma brucei in a semi-defined media. Acta Tropica 36, 289–92.Google Scholar
Clark, S. W. & Meyer, D. I. (1992). Centactin is an actin homologue associated with the centrosome. Nature, London 259, 246–50.CrossRefGoogle Scholar
Dentler, W. L. (1981). Microtubule-membrane interactions in cilia and flagella. International Review of Cytology 72, 1–47.CrossRefGoogle ScholarPubMed
Diano, M., Lebivic, A. & Kirn, M. (1987). A method for the production of highly specific polyclonal antibodies. Analytical Biochemistry 166, 224–9.CrossRefGoogle ScholarPubMed
Fawcett, D. W. (1975). The mammalian spermatozoon. Developmental Biology 44, 394–436.CrossRefGoogle ScholarPubMed
Harris, H. W. & Lux, S. E. (1980). Structural characterization of phosphorylation sites of human erythrocyte spectrin. Journal of Biological Chemistry 255, 11512–20.CrossRefGoogle ScholarPubMed
Hemphill, A., Affolter, M. & Seebeck, T. (1992). A novel microtubule binding motif identified in a high molecular weight microtubule associated protein from Trypanosoma brucei. Journal of Cell Biology 117, 95–103.CrossRefGoogle Scholar
Lafaille, J. J., Linss, J., Krieger, M. A., Souto-Padron, T., Desouza, W. & Goldenberg, S. (1989). Structure and expression of two Trypanosoma cruzi genes encoding antigenic proteins bearing repetitive epitopes. Molecular and Biochemical Parasitology 35, 127–36.CrossRefGoogle ScholarPubMed
Lees-Miller, J. P., Helfman, D. M. & Schroer, T. A. (1992). A vertebrate actin-related protein is a component of a multisubunit complex involved in microtubule based vesicle motility. Nature, London 359, 244–6.CrossRefGoogle ScholarPubMed
Luca, F. C., Bloom, G. S. & Vallee, R. B. (1986). A monoclonal antibody that cross-reacts with phosphorylated epitopes on two microtubule-associated proteins and two neurofilament polypeptides. Proceedings of the National Academy of Sciences, USA 83, 1006–10.CrossRefGoogle ScholarPubMed
MacRae, T. H. & Gull, K. (1990). Purification and assembly in vitro of tubulin from Trypanosoma brucei brucei. The Biochemical Journal 265, 87–93.CrossRefGoogle ScholarPubMed
MacRae, T. H., Lange, B. & Gull, K. (1990). Production and characterisation of monoclonal antibodies to the mammalian sperm cytoskeleton. Molecular Reproduction and Development 25, 384–92.CrossRefGoogle Scholar
Muller, N., Hemphill, A., Imboden, M., Duvallet, G., Dwinger, R. H. & Seebeck, T. (1992). Identification and characterization of two repetitive non-variable antigens from African trypanosomes which are recognized early during infection. Parasitology 104, 111–20.CrossRefGoogle ScholarPubMed
Muller, N., Imboden, M., Detmer, E., Mansfield, J. M. & Seebeck, T. (1993). Cytoskeleton-associated antigens from African trypanosomes are recognized by self-reactive antibodies of uninfected mice. Parasitology 107, 411–17.CrossRefGoogle ScholarPubMed
Rindisbacher, L., Hemphill, A. & Seebeck, T. (1993). A repetitive protein from Trypanosoma brucei which caps the microtubules at the posterior end of the cytoskeleton. Molecular and Biochemical Parasitology 58, 83–96.CrossRefGoogle ScholarPubMed
Russell, D. G., Nevvsam, R. J., Palmer, G. C. N. & Gull, K. (1983). Structural and biochemical characterization of the paraflagellar rod structure of Crithidia fasciculata. European Journal of Cell Biology 30, 137–43.Google ScholarPubMed
Sasse, R. & Gull, K. (1988). Tubulin post-translational modifications and the construction of microtubular organelles in Trypanosoma brucei. Journal of Cell Science 90, 577–89.CrossRefGoogle ScholarPubMed
Schlaeppi, K., Deflorin, J. & Seebeck, T. (1989). The major component of the paraflagellar rod of Trypanosoma brucei is a helical protein that is encoded by two identical, tandemly linked genes. Journal of Cell Biology 109, 1695–709.CrossRefGoogle ScholarPubMed
Schneider, A., Sherwin, T., Sasse, R., Russell, D., Gull, K. & Seebeck, T. (1987). Sub-pellicular and flagellar microtubules of Trypanosoma brucei brucei contain the same alpha tubulin isoforms. Journal of Cell Biology 104, 431–8.CrossRefGoogle Scholar
Sherwin, T. & Gull, K. (1989). The cell division cycle of Trypanosoma brucei brucei: timing of event markers and cytoskeletal modifications. Philosophical Transactions of the Royal Society 323, 573–88.Google Scholar
Sherwin, T., Schneider, A., Sasse, R., Seebeck, T. & Gull, K. (1987). Distinct localization and cell cycle dependence of COOH terminally tyrosinated alpha tubulin in the microtubules of Trypanosoma brucei brucei. Journal of Cell Biology 104, 439–46.CrossRefGoogle Scholar
Towbin, H., Staehelin, T. & Gordon, J. (1979). Electrophoretic transfer from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proceedings of the National Academy of Sciences, USA 76, 4350–4.CrossRefGoogle ScholarPubMed
Woods, A., Sherwin, T., Sasse, R., MacRae, T. H., Baines, A. J. & Gull, K. (1989). Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies. Journal of Cell Science 93, 491–500.CrossRefGoogle ScholarPubMed
Woodward, R., Carden, M. J. & Gull, K. (1994). Molecular characterisation of a novel, repetitive protein of the paraflagellar rod in Trypanosoma brucei. Molecular and Biochemical Parasitology 67, 31–9.CrossRefGoogle Scholar
Woodward, R. & Gull, K. (1990). Timing of nuclear and kinetoplast DNA replication and early morphological events in the cell cycle of Trypanosoma brucei. Journal of Cell Science 95, 49–57.CrossRefGoogle ScholarPubMed