Abstract
Sensory cilia are assembled and maintained by kinesin-2-dependent intraflagellar transport (IFT). We investigated whether two Caenorhabditis elegans α - and β-tubulin isotypes, identified through mutants that lack their cilium distal segments, are delivered to their assembly sites by IFT. Mutations in conserved residues in both tubulins destabilize distal singlet microtubules. One isotype, TBB-4, assembles into microtubules at the tips of the axoneme core and distal segments, where the microtubule tip tracker EB1 is found, and localizes all along the cilium, whereas the other, TBA-5, concentrates in distal singlets. IFT assays, fluorescence recovery after photobleaching analysis and modelling indicate that the continual transport of sub-stoichiometric numbers of these tubulin subunits by the IFT machinery can maintain sensory cilia at their steady-state length.
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Acknowledgements
We thank X. Zhang (Heyer Laboratory), A. Desai and G. Ou for valuable discussion; M. Shohei (Tokyo Women’s Medical University, School of Medicine, Tokyo, Japan) for the deletion mutants, tm2355, tm2394 and tm4200; the Caenorhabditis Genetics Center (funded by the NIH National Center for Research Resources) for strains; and Y. Kohara, Center for Genetic Resource Information, National Institute of Genetics, Japan for EST clones. This work was supported by NIH grant nos GM50718 to J.M.S. and R01NS064273 to S.S.
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J.M.S is the principal investigator of the grant and laboratory that support this IFT project. L.H. and J.M.S. designed the experiments and drafted the manuscript. J.M.S. wrote the manuscript. L.H. carried out most of the experiments. M.T. characterized the qj14 mutant by crossing all of the used IFT markers into it, made the TBB-4::YFP transgenic worms and carried out the EBP-2 experiments. I.B-M. implemented the stochastic tubulin transport and dynamics model and the in silico FRAP model and analysed the results, and helped with the FRAP experiment and transport assay of TBB-4::YFP and analysis of the results. G.C-S. designed and wrote the stochastic tubulin transport and dynamics model and the in silico FRAP model scripts. Y.L. and S.S. carried out the electron microscopy studies and analysed the results. S.A. and B.P. carried out the Y2H assays. All of the authors read the manuscript.
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Hao, L., Thein, M., Brust-Mascher, I. et al. Intraflagellar transport delivers tubulin isotypes to sensory cilium middle and distal segments. Nat Cell Biol 13, 790–798 (2011). https://doi.org/10.1038/ncb2268
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DOI: https://doi.org/10.1038/ncb2268
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