BACKGROUND: The function of the cortical microtubules, composed of alphabeta-tubulin heterodimers, is linked to their organizational state which is subject to spatial and temporal modulation by environmental cues. The role of tubulin posttranslational modifications in these processes is largely unknown. Although antibodies against small tubulin regions represent useful tool for studying molecular configuration of microtubules, data on the exposure of tubulin epitopes on plant microtubules are still limited. RESULTS: Using homology modeling we have generated an Arabidopsis thaliana microtubule protofilament model that served for the prediction of surface exposure of five beta-tubulin epitopes as well as tyrosine residues. Peptide scans newly disclosed the position of epitopes detected by antibodies 18D6 (beta1-10), TUB2.1 (beta426-435) and TU-14 (beta436-445). Experimental verification of the results by immunofluorescence microscopy revealed that the exposure of epitopes depended on the mode of fixation. Moreover, homology modeling showed that only tyrosines in the C-terminal region of beta-tubulins (behind beta425) were exposed on the microtubule external side. Immunofluorescence microscopy revealed tyrosine phosphorylation of microtubules in plant cells, implying that beta-tubulins could be one of the targets for tyrosine kinases. CONCLUSIONS: We predicted surface exposure of five beta-tubulin epitopes, as well as tyrosine residues, on the surface of A. thaliana microtubule protofilament model, and validated the obtained results by immunofluorescence microscopy on cortical microtubules in cells.The results suggest that prediction of epitope exposure on microtubules by means of homology modeling combined with site-directed antibodies can contribute to a better understanding of the interactions of plant microtubules with associated proteins.

• MeSH
• Arabidopsis imunologie   MeSH
• epitopy imunologie   MeSH
• fluorescenční mikroskopie MeSH
• mapování epitopu metody   MeSH
• mikrotubuly imunologie   MeSH
• molekulární modely MeSH
• monoklonální protilátky imunologie   MeSH
• proteiny huseníčku imunologie   MeSH
• tubulin imunologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• epitopy MeSH
• monoklonální protilátky MeSH
• proteiny huseníčku MeSH
• tubulin MeSH

Many of the highly organized microtubular arrangements in ciliates are located in the cortical area containing membrane vesicles and vacuoles. In Tetrahymena thermophila and Paramecium caudatum, immunofluorescence microscopy with the monoclonal antibody TU-06, directed against beta-tubulin, revealed distinct staining of this cortical region alone, while the cilia and other microtubular structures were unstained. The specificity of the antibody was confirmed by immunoblotting and by preabsorption of the antibody with purified tubulin. Double-label immunofluorescence with antibodies against gamma-tubulin, detyrosinated alpha-tubulin, and centrin showed that the TU-06 epitope is localized outside the basal body region. This was also confirmed by immunogold electron microscopy of thin sections. Proteolytic digestion of porcine brain beta-tubulin combined with a peptide scan of immobilized, overlapping peptides disclosed that the epitope was in the beta-tubulin region beta81-95, a region which is phylogenetically highly conserved. As known posttranslational modifications of beta-tubulin are located outside this area, the observed staining pattern cannot be interpreted as evidence of subcellular sequestration of modified tubulin. The limited distribution of the epitope could rather reflect the dependence of TU-06 epitope exposition on conformations of tubulin molecules in microtubule arrangements or on differential masking by interacting proteins.

• MeSH
• buněčná membrána imunologie   MeSH
• buňky 3T3 MeSH
• epitopy analýza   imunologie   metabolismus   MeSH
• imunoblotting MeSH
• mapování epitopu MeSH
• myši MeSH
• Paramecium imunologie   MeSH
• Tetrahymena thermophila imunologie   MeSH
• tubulin imunologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• epitopy MeSH
• tubulin MeSH