The regulation of microtubule dynamics is crucial to many cellular processes. Post-translational tubulin modifications such as tubulin glutamylation are instrumental in regulating microtubule function and influence cellular processes such as centriole stability and neuronal and cilia functions. The recent demonstration that the tubulin tyrosine ligase like (TTLL) proteins are responsible for tubulin glutamylation opens the opportunity for the first in vivo analysis of the role of glutamylation. We are studying the function of glutamylation in cell division and development by analysis of mutant/RNAi phenotypes after disruption of the glutamylases in C. elegans. Immunostaining of worm embryos with an antibody specific for polyglutamylated tubulin reveals the presence of this modification on microtubules. We have obtained a small internal deletion allele of both ttll-4 and ttll-5, which encode the enzymes required to initiate the addition of a polyglutamate side chain to tubulin. Worm strains carrying one or both deletion alleles appear wild type, and by quantitative immunoblotting lack detectable levels of polyglutamylated tubulin. These data indicate that under normal growth conditions, polyglutamylation is not essential for viability. We have also found that that polyglutamylation is not needed under extreme growth conditions (high or low temperature) or for nonessential functions such as the formation of sensory cilia. Furthermore, we have found that microtubule dynamics and centriole stability are apparently normal in the ttll-4;ttll-5 double mutant. We have also developed a reliable strategy for purifiying tubulin from double mutant embryos and plan to use mass spectrometry to confirm the absence of tubulin polyglutamylation in this strain. We have obtained and confirmed deletion alleles of the three other ttll genes. All of these deletion mutants appear wild type. Thus we attempting to construct a worm strain with a deletion in all five ttll genes. This strain will also be subject to the same analysis as described above. So far we have manages to construct several strains in which two or more of the ttll genes have been knocked out. While these worms are viable, they exhibit a male mating defect. Male mating requires functional cilia and thus these results indicate that loss of ttll gene function perturbs cilia function. We are currently in the process of analyzing cilia directly in these mutants. Finally, we have constructed a set of worm strains expressing wild-type and non-glutamylatable versions of beta-tubulin. Our plan is to knockout endogenous tubulin in these lines and determine if the various forms of tubulin can support essential microtubule-based processes.
