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 immunology   MeSH
• Epitopes immunology   MeSH
• Microscopy, Fluorescence MeSH
• Epitope Mapping methods   MeSH
• Microtubules immunology   MeSH
• Models, Molecular MeSH
• Antibodies, Monoclonal immunology   MeSH
• Arabidopsis Proteins immunology   MeSH
• Tubulin immunology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Epitopes MeSH
• Antibodies, Monoclonal MeSH
• Arabidopsis Proteins MeSH
• Tubulin MeSH

Neither the molecular mechanism by which plant microtubules nucleate in the cytoplasm nor the organization of plant mitotic spindles, which lack centrosomes, is well understood. Here, using immunolocalization and cell fractionation techniques, we provide evidence that gamma-tubulin, a universal component of microtubule organizing centers, is present in both the cytoplasm and the nucleus of plant cells. The amount of gamma-tubulin in nuclei increased during the G(2) phase, when cells are synchronized or sorted for particular phases of the cell cycle. gamma-Tubulin appeared on prekinetochores before preprophase arrest caused by inhibition of the cyclin-dependent kinase and before prekinetochore labeling of the mitosis-specific phosphoepitope MPM2. The association of nuclear gamma-tubulin with chromatin displayed moderately strong affinity, as shown by its release after DNase treatment and by using extraction experiments. Subcellular compartmentalization of gamma-tubulin might be an important factor in the organization of plant-specific microtubule arrays and acentriolar mitotic spindles.

• MeSH
• Cell Nucleus chemistry   MeSH
• Cell Cycle MeSH
• Centrioles MeSH
• Fabaceae chemistry   metabolism   MeSH
• Fluorescent Antibody Technique MeSH
• Microscopy, Confocal MeSH
• Plants, Medicinal MeSH
• Mitosis * MeSH
• Plants chemistry   metabolism   MeSH
• Tubulin metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Tubulin MeSH