Investigating the role of TBC1D19 in microtubule dynamics, ciliation and organelle morphology

PROJECT SUMMARY Primary cilia are evolutionarily conserved cellular antennas comprised of a microtubular (MT) axoneme enveloped by a lipid sheath. They are found at the surface of most mammalian cells that have terminally differentiated or entered quiescence. Interestingly, cilia are enriched for proteins required to transduce Sonic Hedgehog and Wnt signaling. Indeed, defects in cilia have been linked to both developmental disorders and cancer, reflecting its intimate relationship with particular signaling pathways that control cell cycle and polarization. Ciliogenesis requires the coordination of multiple events: (1) timely recruitment of various Rab GTPase-positive vesicles to the mother centriole; (2) basal body migration to the plasma membrane; and (3) growth of microtubules to assemble the ciliary axoneme. MT dynamics are controlled by post translational modifications (PTMs), such as glutamylation, and the ciliary axoneme is highly poly-glutamylated. Poly- glutamylation is mediated by a family of tubulin tyrosine-like ligases (TTLLs), including TTLL1, which the Dynlacht laboratory has extensively characterized. Unlike other TTLLs, TTLL1 forms a multimeric Tubulin Poly- glutamylation Complex (TPGC) with seven other proteins. We recently identified TBC1D19, a protein of unknown function, as a novel component of TPGC. TBC1D19 may be an enzyme, as it has a TBC domain, which shares homology to GTP-activating protein (GAP) domains. We generated a knock-out of TBC1D19 in mammalian cells and observed a number of novel and exciting phenotypes, including loss of ciliation and changes in Golgi size and morphology. We will investigate whether these phenotypes can be ascribed to defects in poly-glutamylation, loss of GAP function, or an unknown activity. These findings suggest a potentially wider role for TBC1D19 and the TPGC that links microtubule poly-glutamylation with vesicle trafficking, organelle morphology, and microtubule dynamics. This research will ultimately further our understanding of microtubules and ciliation in development and disease.