Harrison, Alistair; (1993) Microtubules in the ovarioles of notonecta and oncopeltus. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Antibodies specific for tyrosinated, detyrosinated and acetylated tubulin were used to investigate the content and distribution of post-translational modifications of tubulin in the ovarioles of Notonecta glauca glauca and Oncopeltus fasciatus. Furthermore, antibodies were raised against tubulin purified from Notonecta ovarioles and characterised on a number of different cellular systems. Using one- and two-dimensional polyacrylamide gel electrophoresis and Western blotting, ovariole extracts were probed with the isotype specific antibodies. This revealed a major and a minor α-tubulin species in Notonecta, both of which were detyrosinated and acetylated. In contrast, Oncopeltus had only one major α-tubulin species which was tyrosinated. The in situ localisation of tyrosinated tubulin in the nutritive tubes of Notonecta ovarioles revealed a restricted distribution at the inner edge of the periphery of the functional tubes only, this distribution suggesting a novel model by which microtubules are inserted and grow in maturing tubes. Detyrosinated and acetylated tubulin were more widespread, having distributions in both the functional and redundant tubes. In Oncopeltus, tyrosinated tubulin could be visualised in all nutritive tubes, detyrosinated and acetylated tubulin being almost totally absent. This lack of post-translational modifications in Oncopeltus was shown to be due to either the lack or near total inactivation of the enzymatic machinery. The production of monoclonal antibodies against tubulin purified from Notonecta ovarioles was a constantly evolving process, each change producing improved results. In the final production one cloned serum contained antibodies specific for a 114kD epitope, suggesting the presence of a kinesin specific antibody, while 4 sera recognised a 54kD epitope, suggesting the presence of α-tubulin specific antibodies. Further characterisation of the sera produced confusing results which suggested the presence of several different antibody producing hybridomas in each clone and, hence, a conclusive analysis of the antibody content of the sera could not be reached.

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