Abstract
Microtubules are composed of heterodimers of a and ß tubulin. The main differences in amino acid sequence of the seven ß tubulin isotypes occur at the C- terminus, which has been implicated as a key player in microtubule functions and structure. In Drosophila, the C-terminal axonemal motif “EGEFXXX” (where X is either aspartic, D, or glutamic, E, acid) has been hypothesized to be essential for ciliary function and axonemal assembly [1], The axonemal motif is present in some, but not all mammalian ß tubulins. The hypothesis tested here is that this sequence (EGEFEEE) is essential for ciliary function and may thus ascribe a functional reason for the differential synthesis pattern of ß tubulin isotypes.|In isolated reactivated bovine tracheal cilia, monoclonal antibodies directed against the C-terminus of ßi, ßiv, and ßv tubulin blocked ciliary beating in a concentration-dependent manner. Antibodies against the C-termini of other ß tubulin isotypes were ineffective, as were antibodies against other epitopes of ß tubulin or against a tubulin, including the C-terminus of a tubulin. To identify the C-terminal sequence of ß tubulin required for ciliary function, the effect of peptides containing various C-terminal amino acid sequences on ciliary beating of isolated reactivated bovine tracheal cilia was determined. Peptides consisting of the axonemal motif, present in the C-termini of some ß tubulin isotypes, blocked axonemal beating, providing strong evidence of the involvement of this sequence in ciliary beating. Evidence that the C-terminus of a tubulin is not directly involved in ciliary beating was obtained, in addition to defining the sequence of ß tubulin required for ciliary function. Furthermore, ATPase activity of dynein was found not to be influenced by antibodies or peptides in the same way as ciliary beating. The functional domains of the dynein heavy chain were revisited in the literature. In addition to the different tubulin genes, further variations in tubulin expression can be attributed to posttranslational modifications of tubulins. The distribution of the posttranslational modification polyglycylation in cilia and its influence on ciliary beating was determined. For an in vitro analysis, tracheal epithelial cultures expressing beating cilia and organotypic cultures of the organ of Corti were established and are ready for adenoviral transfection with a custom-constructed plasmid.|These results suggest that the axonemal motif sequences of ßi, ßiv, and ßv tubulin are essential for axonemal function, as well as the posttranslational modification polyglycylation. The ATPase active site of dynein is functionally dissociated from the tubulin binding site as previously established on a molecular basis. With this work, a major contribution was made to understanding the evolution of different ß tubulin isotypes in mammals compared to the few isotypes present in protists.