Environmental responsiveness of tubulin glutamylation in sensory cilia is regulated by the p38 MAPK pathway.

Glutamylation is a post-translational modification found on tubulin that can alter the interaction between microtubules (MTs) and associated proteins. The molecular mechanisms regulating tubulin glutamylation in response to the environment are not well understood. Here, we show that in the sensory cilia of Caenorhabditis elegans, tubulin glutamylation is upregulated in response to various signals such as temperature, osmolality, and dietary conditions. Similarly, tubulin glutamylation is modified in mammalian photoreceptor cells following light adaptation. A tubulin glutamate ligase gene ttll-4, which is essential for tubulin glutamylation of axonemal MTs in sensory cilia, is activated by p38 MAPK. Amino acid substitution of TTLL-4 has revealed that a Thr residue (a putative MAPK-phosphorylation site) is required for enhancement of tubulin glutamylation. Intraflagellar transport (IFT), a bidirectional trafficking system specifically observed along axonemal MTs, is required for the formation, maintenance, and function of sensory cilia. Measurement of the velocity of IFT particles revealed that starvation accelerates IFT, which was also dependent on the Thr residue of TTLL-4. Similarly, starvation-induced attenuation of avoidance behaviour from high osmolality conditions was also dependent on ttll-4. Our data suggest that a novel evolutionarily conserved regulatory system exists for tubulin glutamylation in sensory cilia in response to the environment.

Identification of tubulin deglutamylase among Caenorhabditis elegans and mammalian cytosolic carboxypeptidases (CCPs).

Tubulin polyglutamylation is a reversible post-translational modification, serving important roles in microtubule (MT)-related processes. Polyglutamylases of the tubulin tyrosine ligase-like (TTLL) family add glutamate moieties to specific tubulin glutamate residues, whereas as yet unknown deglutamylases shorten polyglutamate chains. First we investigated regulatory machinery of tubulin glutamylation in MT-based sensory cilia of the roundworm Caenorhabditis elegans. We found that ciliary MTs were polyglutamylated by a process requiring ttll-4. Conversely, loss of ccpp-6 gene function, which encodes one of two cytosolic carboxypeptidases (CCPs), resulted in elevated levels of ciliary MT polyglutamylation. Consistent with a deglutamylase function for ccpp-6, overexpression of this gene in ciliated cells decreased polyglutamylation signals. Similarly, we confirmed that overexpression of murine CCP5, one of two sequence orthologs of nematode ccpp-6, caused a dramatic loss of MT polyglutamylation in cultured mammalian cells. Finally, using an in vitro assay for tubulin glutamylation, we found that recombinantly expressed Myc-tagged CCP5 exhibited deglutamylase biochemical activities. Together, these data from two evolutionarily divergent systems identify C. elegans CCPP-6 and its mammalian ortholog CCP5 as a tubulin deglutamylase.

Intraflagellar transport: from molecular characterisation to mechanism.

Research from a wide range of model systems such as Chlamydomonas, C. elegans and mice have shown that intraflagellar transport (IFT) is a bidirectional motility of large protein complexes along cilia and flagella that is essential for building and maintaining these organelles. Since its discovery in 1993, much progress has been made in uncovering the molecular and functional basis of IFT. Presently, many components of the core IFT machinery are known, including the anterograde kinesin 2 motor(s), the IFT-dynein retrograde motor and the collection of at least 17 proteins that makes up the IFT particle. Most significantly, discoveries linking IFT to polycystic kidney disease and other developmental phenotypes have broadened the context of IFT research by demonstrating that primary cilia and IFT are required for processes such as kidney tubule and retinal tissue development, limb bud morphogenesis and organ patterning. Central to the functional basis of IFT is its ability to traffic various ciliary protein cargos, which include structural ciliary subunits, as well as non-structural proteins such as transmembrane channels/receptors and sensory signalling molecules. Indeed, exciting data over the past 3-4 years, linking IFT and primary cilia to developmental and growth factor signalling, as well as the cell cycle, indicates that the current repertoire of IFT cargos is likely to expand. Here we present a comprehensive review of IFT, with particular emphasis on its molecular composition and mechanism of action.