Intraflagellar transport (IFT) trains, multimegadalton assemblies of IFT proteins and motors, traffic proteins in cilia. To study how trains assemble, we employed fluorescence protein-tagged IFT proteins in Chlamydomonas reinhardtii. IFT-A and motor proteins are recruited from the cell body to the basal body pool, assembled into trains, move through the cilium, and disperse back into the cell body. In contrast to this ‘open’ system, IFT-B proteins from retrograde trains reenter the pool and a portion is reused directly in anterograde trains indicating a ‘semi-open’ system. Similar IFT systems were also observed in Tetrahymena thermophila and IMCD3 cells. FRAP analysis indicated that IFT proteins and motors of a given train are sequentially recruited to the basal bodies. IFT dynein and tubulin cargoes are loaded briefly before the trains depart. We conclude that the pool contains IFT trains in multiple stages of assembly queuing for successive release into the cilium upon completion.DOI: http://dx.doi.org/10.7554/eLife.26609.001

The Hedgehog (Hh) signaling pathway plays a key role in cell fate specification, proliferation, and survival during mammalian development. Cells require a small organelle, the primary cilium, to respond properly to Hh signals and the key regulators of Hh signal transduction exhibit dynamic localization to this organelle when the pathway is activated. Here, we investigate the role of Cell Cycle Related kinase (CCRK) in regulation of cilium-dependent Hh signaling in the mouse. Mice mutant for Ccrk exhibit a variety of developmental defects indicative of inappropriate regulation of this pathway. Cell biological, biochemical and genetic analyses indicate that CCRK is required to control the Hedgehog pathway at the level or downstream of Smoothened and upstream of the Gli transcription factors, Gli2 and Gli3. In vitro experiments indicate that Ccrk mutant cells show a greater deficit in response to signaling over long time periods than over short ones. Similar to Chlamydomonas mutants lacking the CCRK homolog, LF2, mouse Ccrk mutant cells show defective regulation of ciliary length and morphology. Ccrk mutant cells exhibit defects in intraflagellar transport (the transport mechanism used to assemble cilia), as well as slowed kinetics of ciliary enrichment of key Hh pathway regulators. Collectively, the data suggest that CCRK positively regulates the kinetics by which ciliary proteins such as Smoothened and Gli2 are imported into the cilium, and that the efficiency of ciliary recruitment allows for potent responses to Hedgehog signaling over long time periods.

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Ciliary microtubules are subject to post-translational modifications that act as a “Tubulin Code” to regulate motor traffic, binding proteins and stability. In humans, loss of CCP1, a cytosolic carboxypeptidase and tubulin deglutamylating enzyme, causes infantile-onset neurodegeneration. In C . elegans , mutations in ccpp-1 , the homolog of CCP1, result in progressive degeneration of neuronal cilia and loss of neuronal function. To identify genes that regulate microtubule glutamylation and ciliary integrity, we performed a forward genetic screen for suppressors of ciliary degeneration in ccpp-1 mutants. We isolated the ttll-5(my38) suppressor, a mutation in a tubulin tyrosine ligase-like glutamylase gene. We show that mutation in the ttll-4 , ttll-5 , or ttll-11 gene suppressed the hyperglutamylation-induced loss of ciliary dye filling and kinesin-2 mislocalization in ccpp-1 cilia. We also identified the nekl-4(my31) suppressor, an allele affecting the NIMA (Never in Mitosis A)-related kinase NEKL-4/NEK10. In humans, NEK10 mutation causes bronchiectasis, an airway and mucociliary transport disorder caused by defective motile cilia. C . elegans NEKL-4 localizes to the ciliary base but does not localize to cilia, suggesting an indirect role in ciliary processes. This work defines a pathway in which glutamylation, a component of the Tubulin Code, is written by TTLL-4, TTLL-5, and TTLL-11; is erased by CCPP-1; is read by ciliary kinesins; and its downstream effects are modulated by NEKL-4 activity. Identification of regulators of microtubule glutamylation in diverse cellular contexts is important to the development of effective therapies for disorders characterized by changes in microtubule glutamylation. By identifying C . elegans genes important for neuronal and ciliary stability, our work may inform research into the roles of the tubulin code in human ciliopathies and neurodegenerative diseases.