Localization of β_V tubulin in the cochlea and cultured cells with a novel monoclonal antibody

Tubulin, the dimeric structural protein of microtubules, is a heterodimer of α and β subunits; both α and β exist as numerous isotypes encoded by different genes. In vertebrates the sequence differences among the β_I, β_II, β_III, β_IV and βV isotypes are highly conserved in evolution, implying that the isotypes may have functional significance. Isotype-specific monoclonal antibodies have been useful in determining the cellular and sub-cellular distributions and possible functions of the β_I; β_II, β_III, and β_IV isotypes; however, little is known about the β_V isotype. We here report the creation and purification of a monoclonal antibody (SHM.12G11) specific for β_V. The antibody was designed to be specific for the C-terminal sequence EEEINE, which is unique to rodent and chicken β_V. The antibody was found to bind specifically to the C-terminal peptide EEEINE, and does not cross-react with the carboxy-termini of either α-tubulin or the other β-tubulin isotypes. However, the antibody also binds to the peptide EEEVNE, but not to the peptide EEEIDG, corresponding respectively to the C-terminal peptides of bovine and human β_V. Immunofluorescence analysis indicates that β_V is found in microtubules of both the interphase network and the mitotic spindle. In gerbils, β_V also occurs in the cochlea where it is found largely in the specialized cells that are unique in containing bundled microtubules with 15 protofilaments.