Exposure of beta-tubulin regions defined by antibodies on an Arabidopsis thaliana microtubule protofilament model and in the cells

Abstract

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.

Figure 1

Reactivity of TU-14 and TUB 2.1 antibodies with β-tubulin fragments. (A) Schematic representation of the β-tubulin peptide map after formic acid cleavage. The positions of formic acid cleavage sites (formic) and the generated proteolytic fragments (β1, β2 and β3) are shown on the thick line. Fragment sizes after incomplete cleavage are outlined below. (B) Coomassie Blue staining of carboxyamidomethylated tubulin heterodimer (lane 1) and isolated β-tubulin (lane 2). α and β denote positions of tubulin subunits. 7.5% SDS-PAGE. (C) Immunostaining with antibodies to β-tubulin fragments generated by formic acid. Lane 1: protein staining of blotted fragments; lanes 2-6: immunostaining with antibodies 18D6 (marker of β3), TU-06 (marker of β1), TU-12 (marker of β2), TUB 2.1 and TU-14. Positions of β-tubulin fragments are indicated on the right margin. 12.5% SDS-PAGE. Molecular mass markers (in kDa) are indicated on the left of B and C.

Figure 2

Immunostaining of peptide scans covering A. thaliana β-tubulin 1 sequences with monoclonal antibodies. Staining with antibodies 18D6 and TU-06 on peptide scans β1-180. Staining with antibodies TU-12, TUB 2.1 and TU-14 on peptide scans β171-447. Peptide scans were formed by immobilized linear 15-meric peptides with 5 amino acid overlaps. Numbers at the top and bottom denote peptide spots in the upper and lower row of the scan, respectively.

Figure 3

Model of A. thaliana microtubule protofilaments with denoted positions of β-tubulin epitopes. Amino acids exposed on surface of protofilaments are colored for epitopes recognized by monoclonal antibodies 18D6, TU-06, TU-12, TUB 2.1 and TU-14. Outer and inner sides of adjacent protofilaments are depicted. α and β denote positions of tubulin subunits in protofilaments; (+) and (-) mark the orientation of protofilaments.

Figure 4

Immunofluorescence staining of A. thaliana microtubules with antibodies to β-tubulin. Fixed (A, C) or unfixed, detergent-extracted (B, D) preparations of primary root epidermal cells were prepared as described in Material and Methods and stained with monoclonal antibodies 18D6 (A-B) and TU-06 (C-D). Bar, 10 μm.

Figure 5

Immunofluorescence double-label staining of A. thaliana unfixed microtubules with antibodies to β-tubulin and phosphotyrosine. Preparations of primary root epidermal cells, preincubated without (A, C, E) or with (B, D, F) sodium orthovanadate (SO) to inhibit phosphatases were stained with monoclonal antibody TUB 2.1 to β-tubulin (A-B) and polyclonal anti-phosphotyrosine antibody P-Tyr (C-D). Figures A, C, E and B, D, F represent the same field. Superpositions (Merge) are shown in E and F. Bar, 10 μm.