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.
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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.
The authors declare no competing financial interests.
Integrated supplementary information
Single color 3D STORM images of SMO in (A) Ptch1−/− and (B) Wild type MEFs. The STORM images are overlaid with conventional wide-field fluorescence images. Scale bars: 500 nm.
RPGRIP1L contains seven coiled-coil segments and 3 C2 domains, including an RPGR-interacting C2 domain (RID). Patient 15-4 carries RPGRIP1L(Thr615Pro); 4-4 and 4-3 carry RPGRIP1L(Ser659Pro). Each JBTS-affected patient also carries a nonsense mutation affecting the C2-N domain or the C2-C domain. Patient 15-4 carries RPGRIP1L(Gln684X); patients 4-4 and 4-3 carry RPGRIP1L(Arg805X).
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Shi, X., Garcia, G., Van De Weghe, J. et al. Super-resolution microscopy reveals that disruption of ciliary transition-zone architecture causes Joubert syndrome. Nat Cell Biol 19, 1178–1188 (2017). https://doi.org/10.1038/ncb3599
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