α- and β-tubulin C-terminal tails with distinct modifications are crucial for ciliary motility and assembly.

α- and β-tubulin have an unstructured glutamate-rich region at their C-terminal tails (CTT). The function of this region in cilia/flagella is still unclear, except that glutamates in CTT act as the sites for posttranslational modifications that affect ciliary motility. A unicellular alga Chlamydomonas possesses only two a-tubulin genes and two b-tubulin genes, each pair encoding an identical protein. This simple gene organization may enable a complete replacement of the wild-type tubulin with its mutated version. Here, using CRISPR/Cas9, we generated mutants expressing tubulins with modified CTTs. We found that the mutant whose four glutamate residues in the α-tubulin CTT have been replaced by alanine almost completely lacked polyglutamylated tubulin and displayed paralyzed cilia. In contrast, the mutant lacking the glutamate-rich region of the β-tubulin CTT assembled short cilia without the central apparatus. This phenotype is similar to the mutants harboring a mutation in a subunit of katanin, whose function has been shown to depend on the b-tubulin CTT. Therefore, our study reveals distinct and important roles of α- and β-tubulin CTT in the formation and function of cilia.