Abstract
The cilium is a microtubule-based organelle that contains a unique complement of proteins for cell motility and signalling functions. Entry into the ciliary compartment is proposed to be regulated at the base of the cilium1. Recent work demonstrated that components of the nuclear import machinery, including the Ran GTPase and importins, regulate ciliary entry2,3,4. We hypothesized that the ciliary base contains a ciliary pore complex whose molecular nature and selective mechanism are similar to those of the nuclear pore complex. By microinjecting fluorescently labelled dextrans and recombinant proteins of various sizes, we characterize a size-dependent diffusion barrier for the entry of cytoplasmic molecules into primary cilia in mammalian cells. We demonstrate that nucleoporins localize to the base of primary and motile cilia and that microinjection of nucleoporin-function-blocking reagents blocks the ciliary entry of kinesin-2 KIF17 motors. Together, this work demonstrates that the physical and molecular nature of the ciliary pore complex is similar to that of the nuclear pore complex, and further extends functional parallels between nuclear and ciliary import.
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Acknowledgements
This work was supported by National Institutes of Health (NIH) grants R01GM070862 (to K.J.V.) and F32GM089034 (to J.F.D.) and funds from the University of Michigan Center for Organogenesis (to K.J.V. and B.M.). We thank B. Allen, J. Martens, T. Hurd, S. Fan, M. Barr and Verhey laboratory members for discussions of the project. We thank B. Dauer, V. Doye, M. Hetzer, R. Wozniak, U. Kutay, K. Kontani and F. Hildebrandt for reagents and advice. We are grateful to B. Craige (University of Massachusetts Medical School) for sharing his technique of trachea cell extraction and to G. Kreitzer (Weill Medical College of Cornell University) for advice on microinjection. We gratefully acknowledge S. Lentz and the Morphology and Image Analysis Core of the Michigan Diabetes Research and Training Center, funded by NIH grant 5P60 DK-20572.
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H.L.K, J.F.D, T.L.B. and C-J.L. carried out experiments. H.L.K., J.F.D., T.L.B., C-J.L., B.M. and K.J.V. designed experiments. All authors contributed to discussions shaping the investigation. H.L.K. and K.J.V. wrote the manuscript, with all authors providing comments and suggestions. K.J.V. directed the project.
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Kee, H., Dishinger, J., Lynne Blasius, T. et al. A size-exclusion permeability barrier and nucleoporins characterize a ciliary pore complex that regulates transport into cilia. Nat Cell Biol 14, 431–437 (2012). https://doi.org/10.1038/ncb2450
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DOI: https://doi.org/10.1038/ncb2450
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