Article | Published:

Super-resolution microscopy reveals that disruption of ciliary transition-zone architecture causes Joubert syndrome

Nature Cell Biology volume 19, pages 11781188 (2017) | Download Citation

  • An Erratum to this article was published on 31 October 2017

This article has been updated

Abstract

Ciliopathies, including nephronophthisis (NPHP), Meckel syndrome (MKS) and Joubert syndrome (JBTS), can be caused by mutations affecting components of the transition zone, a domain near the base of the cilium that controls the protein composition of its membrane. We defined the three-dimensional arrangement of key proteins in the transition zone using two-colour stochastic optical reconstruction microscopy (STORM). NPHP and MKS complex components form nested rings comprised of nine-fold doublets. JBTS-associated mutations in RPGRIP1L or TCTN2 displace certain transition-zone proteins. Diverse ciliary proteins accumulate at the transition zone in wild-type cells, suggesting that the transition zone is a waypoint for proteins entering and exiting the cilium. JBTS-associated mutations in RPGRIP1L disrupt SMO accumulation at the transition zone and the ciliary localization of SMO. We propose that the disruption of transition-zone architecture in JBTS leads to a failure of SMO to accumulate at the transition zone and cilium, disrupting developmental signalling in JBTS.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Change history

  • 25 September 2017

    In the original version of this Article, the name of author Galo Garcia III was coded wrongly, resulting in it being incorrect when exported to citation databases. This has now been corrected, though no visible changes will be apparent.

References

  1. 1.

    The awesome power of dikaryons for studying flagella and basal bodies in Chlamydomonas reinhardtii. Cytoskeleton (Hoboken) 71, 79–94 (2014).

  2. 2.

    & Basal body assembly in ciliates: the power of numbers. Traffic 10, 461–471 (2009).

  3. 3.

    & A primer on the mouse basal body. Cilia 5, 17 (2016).

  4. 4.

    , , & Human basal body basics. Cilia 5, 13 (2016).

  5. 5.

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

  6. 6.

    & The ciliary necklace. A ciliary membrane specialization. J. Cell Biol. 53, 494–509 (1972).

  7. 7.

    & Ciliopathies—from rare inherited cystic kidney diseases to basic cellular function. Mol. Cell. Pediatr. 2, 8 (2015).

  8. 8.

    , & Unraveling the genetics of Joubert and Meckel-Gruber syndromes. J. Pediatr. Genet. 3, 65–78 (2014).

  9. 9.

    et al. A gene mutated in nephronophthisis and retinitis pigmentosa encodes a novel protein, nephroretinin, conserved in evolution. Am. J. Hum. Genet. 71, 1161–1167 (2002).

  10. 10.

    et al. Mutational analysis of the RPGRIP1L gene in patients with Joubert syndrome and nephronophthisis. Kidney Int. 72, 1520–1526 (2007).

  11. 11.

    et al. Mutations in TMEM231 cause Joubert syndrome in French Canadians. J. Med. Genet. 49, 636–641 (2012).

  12. 12.

    et al. Mutations in the gene encoding the basal body protein RPGRIP1L, a nephrocystin-4 interactor, cause Joubert syndrome. Nat. Genet. 39, 882–888 (2007).

  13. 13.

    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).

  14. 14.

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

  15. 15.

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

  16. 16.

    et al. TMEM231, mutated in orofaciodigital and Meckel syndromes, organizes the ciliary transition zone. J. Cell Biol. 209, 129–142 (2015).

  17. 17.

    et al. The ciliary gene RPGRIP1L is mutated in cerebello-oculo-renal syndrome (Joubert syndrome type B) and Meckel syndrome. Nat. Genet. 39, 875–881 (2007).

  18. 18.

    , , & Familial agenesis of the cerebellar vermis. A syndrome of episodic hyperpnea, abnormal eye movements, ataxia, and retardation. Neurology 19, 813–825 (1969).

  19. 19.

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

  20. 20.

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

  21. 21.

    et al. Formation of the transition zone by Mks5/Rpgrip1L establishes a ciliary zone of exclusion (CIZE) that compartmentalises ciliary signalling proteins and controls PIP2 ciliary abundance. EMBO J. 34, 2537–2556 (2015).

  22. 22.

    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).

  23. 23.

    , & The base of the cilium: roles for transition fibres and the transition zone in ciliary formation, maintenance and compartmentalization. EMBO Rep. 13, 608–618 (2012).

  24. 24.

    et al. NPHP4 controls ciliary trafficking of membrane proteins and large soluble proteins at the transition zone. J. Cell Sci. 127, 4714–4727 (2014).

  25. 25.

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

  26. 26.

    & Scoring a backstage pass: mechanisms of ciliogenesis and ciliary access. J. Cell Biol. 197, 697–709 (2012).

  27. 27.

    et al. Subdiffraction-resolution fluorescence microscopy reveals a domain of the centrosome critical for pericentriolar material organization. Nat. Cell Biol. 14, 1159–1168 (2012).

  28. 28.

    , , , & STED microscopy with optimized labeling density reveals 9-fold arrangement of a centriole protein. Biophys. J. 102, 2926–2935 (2012).

  29. 29.

    et al. Superresolution pattern recognition reveals the architectural map of the ciliary transition zone. Sci. Rep. 5, 14096 (2015).

  30. 30.

    , , & Subdiffraction imaging of centrosomes reveals higher-order organizational features of pericentriolar material. Nat. Cell Biol. 14, 1148–1158 (2012).

  31. 31.

    , , & Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science 317, 1749–1753 (2007).

  32. 32.

    , , & Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810–813 (2008).

  33. 33.

    , , , & The Drosophila smoothened gene encodes a seven-pass membrane protein, a putative receptor for the hedgehog signal. Cell 86, 221–232 (1996).

  34. 34.

    , & Patched1 regulates hedgehog signaling at the primary cilium. Science 317, 372–376 (2007).

  35. 35.

    et al. Vertebrate Smoothened functions at the primary cilium. Nature 437, 1018–1021 (2005).

  36. 36.

    et al. Mutations of the human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome. Cell 85, 841–851 (1996).

  37. 37.

    et al. Human homolog of patched, a candidate gene for the basal cell nevus syndrome. Science 272, 1668–1671 (1996).

  38. 38.

    , , & Altered neural cell fates and medulloblastoma in mouse patched mutants. Science 277, 1109–1113 (1997).

  39. 39.

    et al. Sporadic medulloblastomas contain PTCH mutations. Cancer Res. 57, 842–845 (1997).

  40. 40.

    et al. Single-molecule imaging of Hedgehog pathway protein Smoothened in primary cilia reveals binding events regulated by Patched1. Proc. Natl Acad. Sci. USA 112, 8320–8325 (2015).

  41. 41.

    , , , & Small molecule modulation of Smoothened activity. Proc. Natl Acad. Sci. USA 99, 14071–14076 (2002).

  42. 42.

    et al. Adenylate cyclase regulates elongation of mammalian primary cilia. Exp. Cell Res. 315, 2802–2817 (2009).

  43. 43.

    , , & Type III adenylyl cyclase localizes to primary cilia throughout the adult mouse brain. J. Comp. Neurol. 505, 562–571 (2007).

  44. 44.

    et al. Disruption of the type III adenylyl cyclase gene leads to peripheral and behavioral anosmia in transgenic mice. Neuron 27, 487–497 (2000).

  45. 45.

    et al. Inactivation of the mouse adenylyl cyclase 3 gene disrupts male fertility and spermatozoon function. Mol. Endocrinol. 19, 1277–1290 (2005).

  46. 46.

    et al. Adult type 3 adenylyl cyclase-deficient mice are obese. PLoS ONE 4, e6979 (2009).

  47. 47.

    , , , & Localization of intraflagellar transport protein IFT52 identifies basal body transitional fibers as the docking site for IFT particles. Curr. Biol. 11, 1586–1590 (2001).

  48. 48.

    et al. Hypomorphism for RPGRIP1L, a ciliary gene vicinal to the FTO locus, causes increased adiposity in mice. Cell Metab. 19, 767–779 (2014).

  49. 49.

    et al. The NPHP1 gene deletion associated with juvenile nephronophthisis is present in a subset of individuals with Joubert syndrome. Am. J. Hum. Genet. 75, 82–91 (2004).

  50. 50.

    et al. Mutations in the cilia gene ARL13B lead to the classical form of Joubert syndrome. Am. J. Hum. Genet. 83, 170–179 (2008).

  51. 51.

    et al. Mutations in the AHI1 gene, encoding jouberin, cause Joubert syndrome with cortical polymicrogyria. Am. J. Hum. Genet. 75, 979–987 (2004).

  52. 52.

    et al. Abnormal cerebellar development and axonal decussation due to mutations in AHI1 in Joubert syndrome. Nat. Genet. 36, 1008–1013 (2004).

  53. 53.

    , , , & Ftm is a novel basal body protein of cilia involved in Shh signalling. Development 134, 2569–2577 (2007).

  54. 54.

    , , , & Superresolution imaging of chemical synapses in the brain. Neuron 68, 843–856 (2010).

  55. 55.

    et al. Nanoscale architecture of integrin-based cell adhesions. Nature 468, 580–584 (2010).

  56. 56.

    et al. Nuclear pore scaffold structure analyzed by super-resolution microscopy and particle averaging. Science 341, 655–658 (2013).

  57. 57.

    et al. Conserved genetic interactions between ciliopathy complexes cooperatively support ciliogenesis and ciliary signaling. PLoS Genet. 11, e1005627 (2015).

  58. 58.

    & The primary cilium: a signalling centre during vertebrate development. Nat. Rev. Genet. 11, 331–344 (2010).

  59. 59.

    et al. Mutations in KIF7 link Joubert syndrome with Sonic Hedgehog signaling and microtubule dynamics. J. Clin. Invest. 121, 2662–2667 (2011).

  60. 60.

    et al. Murine Joubert syndrome reveals Hedgehog signaling defects as a potential therapeutic target for nephronophthisis. Proc. Natl Acad. Sci. USA 111, 9893–9898 (2014).

  61. 61.

    & Culture and differentiation of mouse tracheal epithelial cells. Methods Mol. Biol. 945, 123–143 (2013).

  62. 62.

    et al. Multicolor far-field fluorescence nanoscopy through isolated detection of distinct molecular species. Nano Lett. 8, 2463–2468 (2008).

  63. 63.

    Estimation of planar curves, surfaces, and nonplanar space-curves defined by implicit equations with applications to edge and range image segmentation. IEEE Trans. Pattern Anal. Mach. Intell. 13, 1115–1138 (1991).

  64. 64.

    , & Efficient subpixel image registration algorithms. Opt. Lett. 33, 156–158 (2008).

  65. 65.

    , & NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).

  66. 66.

    et al. ModBase, a database of annotated comparative protein structure models and associated resources. Nucleic Acids Res. 42, D336–D346 (2014).

  67. 67.

    & Comparative protein structure modeling using MODELLER. Curr. Protoc. Bioinform. 54, 5.6.1–5.6.37 (2016).

  68. 68.

    , , , & The TOPCONS web server for consensus prediction of membrane protein topology and signal peptides. Nucleic Acids Res. 43, W401–W407 (2015).

  69. 69.

    , , & JPred4: a protein secondary structure prediction server. Nucleic Acids Res. 43, W389–W394 (2015).

Download references

Acknowledgements

Structured illumination microscopy was performed on a Nikon N-SIM system in the UCSF Nikon Imaging Center. We thank H. Liu and J. Schnitzbauer for help setting up the STORM system and J. Schnitzbauer in developing the analysis algorithms. We thank V. Herranz-Pérez and J. M. Garcia-Verdugo for providing electron micrographs. This project is supported by the NIH Director’s New Innovator Award (DP2OD008479) to X.S., R.M. and B.H. and by grants from the NIH (AR054396 and GM095941 to J.F.R., F32GM109714 to G.G.III, and U54HD083091 sub-project 6849 to D.D.) and the Burroughs Wellcome Fund and the Packard Foundation to G.G.III and J.F.R. B.H. is a Chan Zuckerberg Biohub investigator.

Author information

Author notes

    • Xiaoyu Shi
    •  & Galo Garcia III

    These authors contributed equally to this work.

    • Ryan McGorty

    Present address: Department of Physics, University of San Diego, San Diego, California 92110, USA.

Affiliations

  1. Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California 94143, USA

    • Xiaoyu Shi
    • , Ryan McGorty
    •  & Bo Huang
  2. Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California 94143, USA

    • Galo Garcia III
    • , Bo Huang
    •  & Jeremy F. Reiter
  3. Cardiovascular Research Institute, University of California, San Francisco, San Francisco, California 94143, USA

    • Galo Garcia III
    •  & Jeremy F. Reiter
  4. Department of Pediatrics, University of Washington Medical Center, Seattle, Washington 98195, USA

    • Julie C. Van De Weghe
    •  & Dan Doherty
  5. Program in Molecular Medicine, University of Massachusetts Medical School, Biotech II, Suite 213, 373 Plantation Street, Worcester, Massachusetts 01605, USA

    • Gregory J. Pazour
  6. Chan Zuckerberg Biohub, San Francisco, California 94158, USA

    • Bo Huang

Authors

  1. Search for Xiaoyu Shi in:

  2. Search for Galo Garcia in:

  3. Search for Julie C. Van De Weghe in:

  4. Search for Ryan McGorty in:

  5. Search for Gregory J. Pazour in:

  6. Search for Dan Doherty in:

  7. Search for Bo Huang in:

  8. Search for Jeremy F. Reiter in:

Contributions

X.S., G.G.III, B.H. and J.F.R. designed the experiments and wrote the manuscript; X.S. and R.M. built the STORM microscope; G.G.III generated the samples; X.S. and G.G.III performed the STORM imaging experiments; and X.S. analysed the STORM data. G.G.III performed the SIM imaging experiments and analysed the SIM data. J.C.V.D.W. and D.D. collected and genotyped the human fibroblasts and provided feedback on the manuscript. G.J.P. generated the α-NPHP1 antibody.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Bo Huang or Jeremy F. Reiter.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    Supplementary Information

  2. 2.

    Life Sciences Reporting Summary

Excel files

  1. 1.

    Supplementary Table 1

    Supplementary Information

  2. 2.

    Supplementary Table 2

    Supplementary Information

  3. 3.

    Supplementary Table 3

    Supplementary Information

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/ncb3599

Further reading