Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A ciliopathy complex at the transition zone protects the cilia as a privileged membrane domain

Nature Cell Biology volume 14, pages 61–72 (2012)Cite this article

Abstract

Using RNAi screening, proteomics, cell biological and mouse genetics approaches, we have identified a complex of nine proteins, seven of which are disrupted in human ciliopathies. A transmembrane component, TMEM231, localizes to the basal body before and independently of intraflagellar transport in a Septin 2 (Sept2)-regulated fashion. The localizations of TMEM231, B9D1 (B9 domain-containing protein 1) and CC2D2A (coiled-coil and C2 domain-containing protein 2A) at the transition zone are dependent on one another and on Sept2. Disruption of the complex in vitro causes a reduction in cilia formation and a loss of signalling receptors from the remaining cilia. Mouse knockouts of B9D1 and TMEM231 have identical defects in Sonic hedgehog (Shh) signalling and ciliogenesis. Strikingly, disruption of the complex increases the rate of diffusion into the ciliary membrane and the amount of plasma-membrane protein in the cilia. The complex that we have described is essential for normal cilia function and acts as a diffusion barrier to maintain the cilia membrane as a compartmentalized signalling organelle.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Identification of the B9 protein complex and its localization at the transition zone.
Figure 2: The B9 complex is required for receptor localization in the cilia.
Figure 3: The B9 complex is required for receptor localization in the cilia.
Figure 4: The B9 protein complex acts as a ciliary barrier.
Figure 5: The B9 complex is required to restrict non-ciliary-membrane protein from the cilia membrane.
Figure 6: The B9 complex is required for Shh signalling in vivo.
Figure 7: The B9 complex is required for ciliogenesis.
Figure 8: The B9 complex regulates ciliogenesis.

Similar content being viewed by others

References

  1. Huangfu, D. et al. Hedgehog signalling in the mouse requires intraflagellar transport proteins. Nature 426, 83–87 (2003).

    Article  CAS  PubMed  Google Scholar 

  2. Rohatgi, R., Milenkovic, L. & Scott, M. P. Patched1 regulates hedgehog signaling at the primary cilium. Science 317, 372–376 (2007).

    Article  CAS  PubMed  Google Scholar 

  3. Tukachinsky, H., Lopez, L. V. & Salic, A. A mechanism for vertebrate Hedgehog signaling: recruitment to cilia and dissociation of SuFu–Gli protein complexes. J. Cell Biol. 191, 415–428 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  5. Davenport, J. R. & Yoder, B. K. An incredible decade for the primary cilium: a look at a once-forgotten organelle. Am. J. Physiol. Renal. Physiol. 289, F1159–F1169 (2005).

    Article  CAS  PubMed  Google Scholar 

  6. Pan, J., Wang, Q. & Snell, W. J. Cilium-generated signaling and cilia-related disorders. Lab. Invest. 85, 452–463 (2005).

    Article  CAS  PubMed  Google Scholar 

  7. Eley, L., Yates, L. M. & Goodship, J. A. Cilia and disease. Curr. Opin. Genet. Dev. 15, 308–314 (2005).

    Article  CAS  PubMed  Google Scholar 

  8. Hunnicutt, G. R., Kosfiszer, M. G. & Snell, W. J. Cell body and flagellar agglutinins in Chlamydomonas reinhardtii: the cell body plasma membrane is a reservoir for agglutinins whose migration to the flagella is regulated by a functional barrier. J. Cell Biol. 111, 1605–1616 (1990).

    Article  CAS  PubMed  Google Scholar 

  9. Vieira, O. V. et al. FAPP2, cilium formation, and compartmentalization of the apical membrane in polarized Madin–Darby canine kidney (MDCK) cells. Proc. Natl Acad. Sci. USA 103, 18556–18561 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Ringo, D. L. Flagellar motion and fine structure of the flagellar apparatus in Chlamydomonas. J. Cell Biol. 33, 543–571 (1967).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Rohatgi, R. & Snell, W. J. The ciliary membrane. Curr. Opin. Cell Biol. 22, 541–546 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 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  PubMed  PubMed Central  Google Scholar 

  14. 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  PubMed  PubMed Central  Google Scholar 

  15. Hu, Q. et al. A septin diffusion barrier at the base of the primary cilium maintains ciliary membrane protein distribution. Science 329, 436–439 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Ashique, A. M. et al. The Rfx4 transcription factor modulates Shh signaling by regional control of ciliogenesis. Sci. Signal 2, ra70 (2009).

    Article  PubMed  Google Scholar 

  17. Torres, J. Z., Miller, J. J. & Jackson, P. K. High-throughput generation of tagged stable cell lines for proteomic analysis. Proteomics 9, 2888–2891 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Nachury, M. V. Tandem affinity purification of the BBSome, a critical regulator of Rab8 in ciliogenesis. Methods Enzymol. 439, 501–513 (2008).

    Article  CAS  PubMed  Google Scholar 

  19. Cardenas-Rodriguez, M. & Badano, J. L. Ciliary biology: understanding the cellular and genetic basis of human ciliopathies. Am. J. Med. Genet. C Semin. Med. Genet. 151C, 263–280 (2009).

    Article  CAS  PubMed  Google Scholar 

  20. Reiter, J. F. & Skarnes, W. C. Tectonic, a novel regulator of the Hedgehog pathway required for both activation and inhibition. Genes Dev. 20, 22–27 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Hopp, K. et al. B9D1 is revealed as a novel Meckel syndrome (MKS) gene by targeted exon-enriched next-generation sequencing and deletion analysis. Hum. Mol. Genet. 20, 2524–2534 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Dowdle, W. E. et al. Disruption of a ciliary B9 protein complex causes Meckel syndrome. Am. J. Hum. Genet. 89, 94–110 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. 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  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Sorokin, S. P. Reconstructions of centriole formation and ciliogenesis in mammalian lungs. J. Cell Sci. 3, 207–230 (1968).

    CAS  PubMed  Google Scholar 

  26. Pazour, G. J. et al. Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene tg737, are required for assembly of cilia and flagella. J. Cell Biol. 151, 709–718 (2000).

    Article  CAS  PubMed  PubMed Central  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  PubMed  PubMed Central  Google Scholar 

  28. Murcia, N. S. et al. The Oak Ridge Polycystic Kidney (orpk) disease gene is required for left-right axis determination. Development 127, 2347–2355 (2000).

    CAS  PubMed  Google Scholar 

  29. Louie, C. M. et al. AHI1 is required for photoreceptor outer segment development and is a modifier for retinal degeneration in nephronophthisis. Nat. Genet. 42, 175–180 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Bachmann-Gagescu, R. et al. The ciliopathy gene cc2d2a controls zebrafish photoreceptor outer segment development through a role in Rab8-dependent vesicle trafficking. Hum. Mol. Genet. 20, 4041–4055 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Town, T. et al. The stumpy gene is required for mammalian ciliogenesis. Proc. Natl Acad. Sci. USA 105, 2853–2858 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Breunig, J. J. et al. Primary cilia regulate hippocampal neurogenesis by mediating sonic hedgehog signaling. Proc. Natl Acad. Sci. USA 105, 13127–13132 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Weatherbee, S. D., Niswander, L. A. & Anderson, K. V. A mouse model for Meckel syndrome reveals Mks1 is required for ciliogenesis and Hedgehog signaling. Hum. Mol. Genet. 18, 4565–4575 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Cui, C. et al. Disruption of Mks1 localization to the mother centriole causes cilia defects and developmental malformations in Meckel–Gruber syndrome. Dis. Model Mech. 4, 43–56 (2011).

    CAS  PubMed  Google Scholar 

  35. Lancaster, M. A. et al. Impaired Wnt-beta-catenin signaling disrupts adult renal homeostasis and leads to cystic kidney ciliopathy. Nat. Med. 15, 1046–1054 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Williams, C. L., Winkelbauer, M. E., Schafer, J. C., Michaud, E. J. & Yoder, B. K. Functional redundancy of the B9 proteins and nephrocystins in Caenorhabditis elegans ciliogenesis. Mol. Biol. Cell 19, 2154–2168 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Bialas, N. J. et al. Functional interactions between the ciliopathy-associated Meckel syndrome 1 (MKS1) protein and two novel MKS1-related (MKSR) proteins. J. Cell Sci. 122, 611–624 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Williams, C. L., Masyukova, S. V. & Yoder, B. K. Normal ciliogenesis requires synergy between the cystic kidney disease genes MKS-3 and NPHP-4. J. Am. Soc. Nephrol. 21, 782–793 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Gorden, N. T. et al. CC2D2A is mutated in Joubert syndrome and interacts with the ciliopathy-associated basal body protein CEP290. Am. J. Hum. Genet. 83, 559–571 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Fliegauf, M. et al. Nephrocystin specifically localizes to the transition zone of renal and respiratory cilia and photoreceptor connecting cilia. J. Am. Soc. Nephrol. 17, 2424–2433 (2006).

    Article  CAS  PubMed  Google Scholar 

  41. Won, J. et al. NPHP4 is necessary for normal photoreceptor ribbon synapse maintenance and outer segment formation, and for sperm development. Hum. Mol. Genet. 20, 482–496 (2010).

    PubMed  PubMed Central  Google Scholar 

  42. Valente, E. M. et al. Mutations in TMEM216 perturb ciliogenesis and cause Joubert, Meckel and related syndromes. Nat. Genet. 42, 619–625 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Evangelista, M. et al. Kinome siRNA screen identifies regulators of ciliogenesis and hedgehog signal transduction. Sci. Signal 1, ra7 (2008).

    Article  PubMed  Google Scholar 

  44. Tang, T. et al. A mouse knockout library for secreted and transmembrane proteins. Nat. Biotechnol. 28, 749–755 (2010).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank M. Roose-Girma (Genentech) for generating B9D1-knockout mouse; J. Miller, S. Mukhopadhyay and P. Jackson (all Genentech) for various LAP tagging constructs and advice on tandem affinity purification technology; S. Francis (Genentech) for GFP–CEACAM1 and GFP–GPI retroviral particles; B. Bingol for help with ImageJ analysis; D. Grant, M. Solloway and H. Tian for help with histology; J. Ernst and H. Li for octyl-Shh; and the Genentech research community for their advice and assistance. TMEM231-knockout mice were produced in a collaboration between Genentech and Lexicon Pharmaceuticals (The Woodlands) to analyse the function of about 500 secreted and transmembrane proteins. Genentech provided financial support.

Author information

Authors and Affiliations

  1. Department of Molecular Biology, Genentech, South San Francisco, California 94080, USA

    Ben Chih & Andrew S. Peterson

  2. Department of Protein Chemistry, Genentech, South San Francisco, California 94080, USA

    Peter Liu, Yvonne Chinn, Philip E. Hass & Wendy Sandoval

  3. Department of Pathology, Center for Advance Light Microscopy, Genentech, South San Francisco, California 94080, USA

    Cecile Chalouni & Laszlo G. Komuves

Authors
  1. Ben Chih
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Peter Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yvonne Chinn
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Cecile Chalouni
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Laszlo G. Komuves
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Philip E. Hass
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Wendy Sandoval
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Andrew S. Peterson
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

B.C. planned, carried out and analysed experiments. P.L. and W.S. carried out mass spectrometry experiments. Y.C. and P.E.H. collected the gel filtration samples. C.C. and L.G.K acquiredthe super-resolution images. B.C. and A.S.P. designed and interpreted the experiments and wrotethe manuscript.

Corresponding author

Correspondence to Andrew S. Peterson.

Ethics declarations

Competing interests

All authors are employees of Genentech, a for-profit institution.

Supplementary information

Supplementary Information

Supplementary Information (PDF 550 kb)

Supplementary Movie 1

Supplementary Information (AVI 297 kb)

Supplementary Table 1

Supplementary Information (XLS 28 kb)

Supplementary Table 2

Supplementary Information (XLS 30 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chih, B., Liu, P., Chinn, Y. et al. A ciliopathy complex at the transition zone protects the cilia as a privileged membrane domain. Nat Cell Biol 14, 61–72 (2012). https://doi.org/10.1038/ncb2410

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb2410

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing