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A size-exclusion permeability barrier and nucleoporins characterize a ciliary pore complex that regulates transport into cilia

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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Figure 1: The ciliary base acts as a size-dependent barrier for entry of cytoplasmic dextran molecules.
Figure 2: The ciliary base acts as a size-dependent barrier for entry of inert cytoplasmic proteins.
Figure 3: Fluorescently tagged nucleoporins localize to the base of primary cilia.
Figure 4: Endogenous nucleoporins localize to the base of cilia.
Figure 5: Microinjection of nucleoporin-function-blocking reagents into cells restricts the ciliary entry of KIF17 motors.

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References

  1. Rosenbaum, J. L. & Witman, G. B. Intraflagellar transport. Nat. Rev. Mol. Cell Biol. 3, 813–825 (2002).

    Article  CAS  Google Scholar 

  2. Dishinger, J. F. et al. Ciliary entry of the kinesin-2 motor KIF17 is regulated by importin-beta2 and RanGTP. Nat. Cell Biol. 12, 703–710 (2010).

    Article  CAS  Google Scholar 

  3. Fan, S. et al. Induction of Ran GTP drives ciliogenesis. Mol. Biol. Cell 22, 4539–4548 (2011).

    Article  CAS  Google Scholar 

  4. Hurd, T. W., Fan, S. & Margolis, B. L. Localization of retinitis pigmentosa 2 to cilia is regulated by Importin β2. J. Cell Sci. 124, 718–726 (2011).

    Article  CAS  Google Scholar 

  5. Berbari, N. F., O’Connor, A. K., Haycraft, C. J. & Yoder, B. K. The primary cilium as a complex signaling center. Curr. Biol. 19, R526–R535 (2009).

    Article  CAS  Google Scholar 

  6. Satir, P. & Christensen, S. T. Overview of structure and function of mammalian cilia. Annu. Rev. Physiol. 69, 377–400 (2007).

    Article  CAS  Google Scholar 

  7. Badano, J. L., Mitsuma, N., Beales, P. L. & Katsanis, N. The ciliopathies: an emerging class of human genetic disorders. Annu. Rev. Genomics Hum. Genet. 7, 125–148 (2006).

    Article  CAS  Google Scholar 

  8. Sharma, N., Berbari, N. F. & Yoder, B. K. Ciliary dysfunction in developmental abnormalities and diseases. Curr. Top. Dev. Biol. 85, 371–427 (2008).

    Article  CAS  Google Scholar 

  9. Hildebrandt, F., Benzing, T. & Katsanis, N. Ciliopathies. New Engl. J. Med. 364, 1533–1543 (2011).

    Article  CAS  Google Scholar 

  10. Nachury, M. V., Seeley, E. S. & Jin, H. Trafficking to the ciliary membrane: how to get across the periciliary diffusion barrier? Annu. Rev. Cell Dev. Biol. 26, 59–87 (2010).

    Article  CAS  Google Scholar 

  11. Anderson, R. G. The three-dimensional structure of the basal body from the rhesus monkey oviduct. J. Cell Biol. 54, 246–265 (1972).

    Article  CAS  Google Scholar 

  12. Deane, J. A., Cole, D. G., Seeley, E. S., Diener, D. R. & Rosenbaum, J. L. Localization of intraflagellar transport protein IFT52 identifies basal body transitional fibers as the docking site for IFT particles. Curr. Biol. 11, 1586–1590 (2001).

    Article  CAS  Google Scholar 

  13. Gilula, N. B. & Satir, P. The ciliary necklace. A ciliary membrane specialization. J. Cell Biol. 53, 494–509 (1972).

    Article  CAS  Google Scholar 

  14. Craige, B. et al. CEP290 tethers flagellar transition zone microtubules to the membrane and regulates flagellar protein content. J. Cell Biol. 190, 927–940 (2010).

    Article  CAS  Google Scholar 

  15. Garcia-Gonzalo, F. R. et al. A transition zone complex regulates mammalian ciliogenesis and ciliary membrane composition. Nat. Genet. 43, 776–784 (2011).

    Article  CAS  Google Scholar 

  16. Sang, L. et al. Mapping the NPHP–JBTS–MKS protein network reveals ciliopathy disease genes and pathways. Cell 145, 513–528 (2011).

    Article  CAS  Google Scholar 

  17. Williams, C. L. et al. MKS and NPHP modules cooperate to establish basal body/transition zone membrane associations and ciliary gate function during ciliogenesis. J. Cell Biol. 192, 1023–1041 (2011).

    Article  CAS  Google Scholar 

  18. Chih, B. et al. A ciliopathy complex at the transition zone protects the cilia as a privileged membrane domain. Nat. Cell Biol. 14, 61–72 (2012).

    Article  CAS  Google Scholar 

  19. Fahrenkrog, B. & Aebi, U. The nuclear pore complex: nucleocytoplasmic transport and beyond. Nat. Rev. Mol. Cell Biol. 4, 757–766 (2003).

    Article  CAS  Google Scholar 

  20. Gorlich, D. & Kutay, U. Transport between the cell nucleus and the cytoplasm. Annu. Rev. Cell Dev. Biol. 15, 607–660 (1999).

    Article  CAS  Google Scholar 

  21. Stewart, M. Molecular mechanism of the nuclear protein import cycle. Nat. Rev. Mol. Cell Biol. 8, 195–208 (2007).

    Article  CAS  Google Scholar 

  22. Lang, I., Scholz, M. & Peters, R. Molecular mobility and nucleocytoplasmic flux in hepatoma cells. J. Cell Biol. 102, 1183–1190 (1986).

    Article  CAS  Google Scholar 

  23. Paine, P. L., Moore, L. C. & Horowitz, S. B. Nuclear envelope permeability. Nature 254, 109–114 (1975).

    Article  CAS  Google Scholar 

  24. Takao, D. & Kamimura, S. Geometry-specific heterogeneity of the apparent diffusion rate of materials inside sperm cells. Biophys. J. 98, 1582–1588 (2010).

    Article  CAS  Google Scholar 

  25. Mohr, D., Frey, S., Fischer, T., Guttler, T. & Gorlich, D. Characterisation of the passive permeability barrier of nuclear pore complexes. EMBO J. 28, 2541–2553 (2009).

    Article  CAS  Google Scholar 

  26. Calvert, P. D., Schiesser, W. E. & Pugh, E. N. Jr Diffusion of a soluble protein, photoactivatable GFP, through a sensory cilium. J. Gen. Physiol. 135, 173–196 (2010).

    Article  CAS  Google Scholar 

  27. Francis, S. S., Sfakianos, J., Lo, B. & Mellman, I. A hierarchy of signals regulates entry of membrane proteins into the ciliary membrane domain in epithelial cells. J. Cell Biol. 193, 219–233 (2011).

    Article  CAS  Google Scholar 

  28. Brohawn, S. G., Partridge, J. R., Whittle, J. R. & Schwartz, T. U. The nuclear pore complex has entered the atomic age. Structure 17, 1156–1168 (2009).

    Article  CAS  Google Scholar 

  29. D’Angelo, M. A. & Hetzer, M. W. Structure, dynamics and function of nuclear pore complexes. Trends Cell Biol. 18, 456–466 (2008).

    Article  Google Scholar 

  30. Dultz, E. & Ellenberg, J. Live imaging of single nuclear pores reveals unique assembly kinetics and mechanism in interphase. J. Cell Biol. 191, 15–22 (2010).

    Article  CAS  Google Scholar 

  31. Rabut, G., Doye, V. & Ellenberg, J. Mapping the dynamic organization of the nuclear pore complex inside single living cells. Nat. Cell Biol. 6, 1114–1121 (2004).

    Article  CAS  Google Scholar 

  32. Murrell, J. R. & Hunter, D. D. An olfactory sensory neuron line, odora, properly targets olfactory proteins and responds to odorants. J. Neurosci. 19, 8260–8270 (1999).

    Article  CAS  Google Scholar 

  33. Davis, L. I. & Blobel, G. Identification and characterization of a nuclear pore complex protein. Cell 45, 699–709 (1986).

    Article  CAS  Google Scholar 

  34. Otto, E. A. et al. Candidate exome capture identifies mutation of SDCCAG8 as the cause of a retinal–renal ciliopathy. Nat. Genet. 42, 840–850 (2010).

    Article  CAS  Google Scholar 

  35. Clever, J., Yamada, M. & Kasamatsu, H. Import of simian virus 40 virions through nuclear pore complexes. Proc. Natl Acad. Sci. USA 88, 7333–7337 (1991).

    Article  CAS  Google Scholar 

  36. Kutay, U., Izaurralde, E., Bischoff, F. R., Mattaj, I. W. & Gorlich, D. Dominant-negative mutants of importin- β block multiple pathways of import and export through the nuclear pore complex. EMBO J. 16, 1153–1163 (1997).

    Article  CAS  Google Scholar 

  37. Strambio-De-Castillia, C., Niepel, M. & Rout, M. P. The nuclear pore complex: bridging nuclear transport and gene regulation. Nat. Rev. Mol. Cell Biol. 11, 490–501 (2010).

    Article  CAS  Google Scholar 

  38. Walther, T. C. et al. The nucleoporin Nup153 is required for nuclear pore basket formation, nuclear pore complex anchoring and import of a subset of nuclear proteins. EMBO J. 20, 5703–5714 (2001).

    Article  CAS  Google Scholar 

  39. Hammond, J. W., Blasius, T. L., Soppina, V., Cai, D. & Verhey, K. J. Autoinhibition of the kinesin-2 motor KIF17 via dual intramolecular mechanisms. J. Cell Biol. 189, 1013–1025 (2010).

    Article  CAS  Google Scholar 

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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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Correspondence to Kristen J. Verhey.

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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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