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 immunoblotting lack detectable levels of polyglutamylated tubulin. These data indicate that under normal growth conditions, polyglutamylation is not essential for viability. We are now exploring the possibility that polyglutamylation might only be needed under extreme growth conditions (high or low temperature) or for nonessential functions such as the formation of sensory cilia. We are also characterizing the subcellular distribution of TTL-4 and TTLL-5 using affinity purified antibodies and GFP tagging. So far, both approaches indicate that a major fraction of each protein is localized to nuclei. However, a smaller fraction may also be localized to centrosomes.
