Chlamydomonas γ-tubulin mutations reveal a critical role of γ-TuRC in maintaining the stability of centriolar microtubules

The centriolar triplet microtubule consists of an A-tubule with 13 protofilaments, and B- and C-tubules, each with 10 protofilaments. Although the formation of the triplets has been shown to require γ-tubulin, its specific role in the formation of each tubule remains elusive. We isolated two novel Chlamydomonas reinhardtii mutants, bld13-1 and bld13-2 , each expressing γ-tubulin with a single amino-acid substitution (T292I, E89D, respectively). Similar to known centriole-deficient mutants, both mutants exhibited defects in ciliary assembly, nuclear number, and the number and orientation of cytoplasmic microtubules. Genetic analyses of the mutants, along with expression of the mutant γ-tubulins in the wild-type cells, showed that both mutants exert dominant-negative effects over wild-type γ-tubulin. Interestingly, although the centrioles in these mutants retained the typical nine triplet structure, their triplets frequently lacked several protofilaments in specific regions of the A- and C-tubules. The protofilament loss occurs more frequently at the proximal end of the centriole. These structural defects strongly suggest that γ-tubulin is essential for the stability of the A- and C-tubules of centriolar triplets.