Temporal and spatial expression patterns of TUB9, a β-tubulin gene of Arabidopsis thaliana
A panel of nine antibodies, specific to antigenic determinants located on N- or C-terminal structural domains of alpha and beta subunits of animal tubulin, and antibodies against acetylated, tyrosinated and polyglutamylated tubulins were utilized for probing the Nicotiana tabacum microtubules. The specificity of antibodies was confirmed by immunoblotting on whole cell lysates and on tubulin isoforms separated by high-resolution isoelectric focusing. Whereas antibodies TU-01 and TU-09 reacted with all alpha-tubulin isoforms and TU-06 reacted with all beta-tubulin isoforms, the other antibodies reacted with a limited number of tubulin isoforms. Antibody TU-14 reacted only with two beta-tubulin charge variants. In fixed cells, each of the antibodies stained microtubules of preprophase band, mitotic spindle and phragmoplast. Cortical microtubules were stained by all antibodies except TU-02 and TU-03, which did not decorate microtubules in interphase cells. Immunostaining of unfixed detergent-extracted cells revealed that antibodies against determinants on the C-terminal domains of both subunits decorated microtubules, but these were not stained with antibodies to determinants on the N-terminal domains. These data indicate that in plant microtubules at least several parts of the N-terminal domains of both subunits are either not exposed on the microtubule surface or are masked by the other proteins. In contrast, parts of the C-terminal domains are exposed on the exterior of microtubules. As for animal tubulins the majority of posttranslational modifications as well as binding sites for microtubule-associated proteins (MAPs) have been located to these regions, it is possible also in higher plants that the C-terminal structural domains of both tubulin subunits participate in the modulation of tubulin interactions with associated proteins.