Glutamylation is a post-translational modification that occurs on tubulin and regulates tubulin function. Microtubules play a role in cell division, cell motility, and the transportation of vesicles and organelles. A family of enzymes called tubulin tyrosine ligase-like (TTLL) enzymes catalyze the glutamylation of tubulin. Eight TTLL enzymes are encoded within the human genome and mutations of these enzymes has been shown to affect cilia mobility. Individual deletions and reduced function of select TTLL enzymes has been shown to impair the mobility of cilia in various cell types in humans, mice, and Caenorhabditis elegans (C. elegans). C. elegans express six of these eight TTLL enzymes and will be used as a model system to investigate the role of tubulin glutamylation in microtubule function. To examine the role of these enzymes, a strain with mutations in multiple TTLL enzymes was generated (ttll-4, ttll-9, and ttll-11) and viability, brood size, male mating, and male mating efficiency assays were preformed. The gathered information on the triple mutant's viability, brood size, observations of behavior(s), and the confirmed triple mutant C. elegan shall be used in continuing studies on TTLL enzymes in C. elegans. This research will yield further insight to the role(s) of TTLL enzymes and glutamylation in regulating microtubule function.

Polyglutamylation is a post-translational modification (PTM) of tubulin thought to play a role in a number of key microtubule-related functions across eukaryotic life, such as cell motility, spindle function, and neuronal growth. An inability to perform this modification has been linked to neurodegenerative diseases such as Alzheimer’s and Parkinson’s, as well as diseases caused by dysfunctional cilia and spindles. The modification is mediated by the enzyme family tubulin tyrosine ligase-like (TTLL), which act as glutamate ligases on tubulin. The nematode Caenorhabditis elegans expresses five such enzymes, TTLL-4, -5, -9, -11, and -15, compared to 13 predicted mammalian enzymes. Previous research in C. elegans indicates that the TTLL enzymes act redundantly and may be required for proper function of certain neurons in males. In this research, a C. elegans quadruple mutant with deletions of four of the five tubulin glutamylating enzymes (all except TTLL-4) was confirmed via PCR and assayed for male mating efficiency, viability, brood size, osmotic avoidance, and dye-filling (staining) of sensory neurons, which shows their structural integrity. We predict that male mating efficiency will be found to be significantly reduced compared to a wild-type control and similar to a triple mutant, supporting the hypothesis that the TTLL enzymes act redundantly and that multiple enzymes are required for the proper function of male-specific neurons. Viability will be mostly unaffected in terms of reaching adulthood, but mutant worms appear to grow slower than wild-type controls. Brood size, osmotic avoidance, and dye-filling will be similar to wild-type, as with the triple mutant. The results will need to be compared to a quintuple mutant, and indicate that tubulin glutamylation may be a good target for human brain disease research.