Fig 3. C. elegans CEP-290 is a transition zone protein required for ciliary gate (membrane diffusion barrier) function and transition zone assembly.(A) C. elegans CEP-290 (Y47G6A.17) tagged with GFP (green) is expressed only in ciliated sensory neurons (including amphids and phasmids) and isspecifically enriched at the ciliary transition zone (TZ). tdTomato-tagged XBX-1 (red) is an IFT protein that marks the basal body (BB) and axoneme. Scale bars in panels A, C, and D are 4 um. (B)
cep-290 gene structure and likely null allele (
gk415029 nonsense mutation) isolated from the Million Mutation Project (MMP) mutant strain VC30108. (C) CEP-290 is required for maintaining the functional integrity of the TZ. Two membrane-associated proteins (TRAM-1a::tdTomato and RPI-2::GFP) normally present at the base of cilia and not present within cilia show abnormal ciliary entry ("leakage") or accumulation in the
cep-290 mutant (shown as asterisks). MKSR-1::tdTomato and MKS-2::GFP are TZ protein comarkers (in wild-type) that mislocalise in the
cep-290 mutant (dotted ellipse). (D) CEP-290 is required to exclude membrane-associated GFP-tagged ARL-13 (green) from the transition zone and compartmentalise it within the ciliary middle segment (MS), consistent with a role for CEP-290 in TZ gate function. In the
cep-290 mutant, ARL-13 is also seen outside of the cilium in the periciliary membrane compartment (PCMC) region (mislocalisation shown with asterisks). (E) Transition zone ultrastructure is missing in
cep290 mutants. Transmission electron microscopy images from serial cross-sections of the amphid channel (pore) in wild-type and
cep-290 mutant worms. Boxed numbers denote positioning of sections relative to anterior-most section (+0); section positions also shown in schematics on right-hand side. Wild-type amphid channels possess ten ciliary axonemes, each with distal segment (ds; outer singlet A-tubules), middle segment (ms; outer doublet A/B tubules), and transition zone (TZ; outer doublet A/B tubules, Y-links, apical ring) compartments. In
cep-290 mutants, one to two cilia are missing from the distal pore, indicating that one to two axonemes are short. TZ ultrastructure is severely disrupted in
cep-290 worms; most or all Y-link structure is missing, as is the apical ring that draws together outer doublet MTs with varying numbers of inner singlet microtubules. MT doublets are also frequently disorganised near the ciliarybase. Schematics denote the observed ultrastructural phenotypes (only three ciliary axonemes shown for simplicity). pcmc; periciliary membranecompartment. Scale bars, 200 nm (large, low-magnification top panel images) and 100 nm (small, high-magnification top panel images and all bottom panelTZ images).