Localization of post-translationally modified alpha-tubulin and pseudocyst formation in tritrichomonads

The distribution of acetylated tubulin was investigated in members of the Tritrichomonadinae subfamily using a specific monoclonal antibody and indirect immunofluorescence microscopy. Species probed in this study were: Tritrichomonas foetus, T. mobilensis, T. muris, and an unnamed tritrichomonad isolated from the cecum of cotton rats (Sigmodon hispidus). Additionally, the distribution of glutamylated tubulin in T. muris was investigated using the GT 335 antibody to the glutamyl motif. Although acetylated alpha-tubulin was ubiquitously distributed throughout the axostyle and flagella of T. foetus and T. mobilensis, a distinct and unusual labeling pattern was observed in 'T. muris'-type or pseudocyst-forming trichomonads. In trophozoite stages of T. muris and the cotton rat isolate, flagella were intensely labeled, but acetylation of axostylar tubulin appeared to be limited to the anterior and posterior thirds. However, trophozoites labeled with an antibody to alpha-tubulin or glutamylated tubulin demonstrated no such discontinuity in axostylar staining. Additionally, pseudocysts labeled with anti-alpha-tubulin and anti-acetylated alpha-tubulin were all found to possess continuously fluorescent axostyles. That the mid-region axostyle remained unlabeled by anti-acetylated tubulin in trophozoites indicates possible deacetylase activity, which may have functional implications with respect to the life cycle. Glutamylated tubulin appeared to be distributed throughout the axostyle and flagella, which fluoresced brightly in T. muris.