Figure 1. Disruption of
ift-20 results in defective cilium assembly and function in C. elegans: (A) Schematic of the Y110A7A.20 locus, showing the predicted
ift-20 exons (in grey boxes) and the relative position of the X-box motif according to (Blacque et al. 2005; Efimenko et al. 2005). The
ok3191 deletion/substitution allele (-386bp, +2bp=C+T) was mapped and is represented in purple. (B) Examples of the DiI lipophilic dye-filling test in sensory phasmid neurons of wild-type and
ift-20 null worms. 100% of the
ift-20 null worms fail to incorporate dye (N≥15 worms per strain). Scale bar, 10 μm. (C) Examples of the dendritic ends of phasmid neurons from wild-type and
ift-20 mutants expressing GFP::CCEP-290 and mCherry::HYLS-1, labeling the transition zone and the base of cilia, respectively (N≥18 cilia per strain). Scale bar, 2 μm. (D) Illustration of the ciliated dendritic ends of phasmid neurons. (E) Examples of cilia of phasmid neurons of wild-type and
ift-20 mutants, labeled with soluble GFP (sGFP; able to enter cilia) and mCherry::HYLS-1 (ciliary base). *refers to a dendrite from a different type of neuron (PQR) also present in the figure. (F) Examples of the distribution of endogenously labeled IFT-74::GFP and its incorporation into cilia of phasmid neurons. White arrows point at the tips of cilia. Scale bars, 2 μm. (G, H) IFT-20-deficient worms have defects in ciliogenesis but manage to assemble severely shortened cilia as shown in the quantifications (N≥107 sGFP labeled cilia were analyzed per strain; N≥80 cilia with detectable IFT-74::GFP were analyzed per strain). Bars in graphs show the average cilia length (± SD). (I) Average IFT-74::GFP signal distribution and intensity (± SEM) along cilia are altered in
ift-20 null worms, revealing that they incorporate less IFT-74 (N≥80 cilia for each strain; worms with no ciliary IFT-74 signal were not included). (J) Osmotic avoidance assay shows that sensory cilia of
ift-20 null worms fail to sense the high glycerol concentrations of the ring surrounding them, and cross it to escape. (K) Graph shows the average percentage of worms (± SD) that cannot sense a hypertonic glycerol barrier (N=28 worms in controls; N=35
ift-20 null worms; analyzed over 11 independent experiments). (L) Mating assay was also performed to test the proficiency of sensory cilia functions in
ift-20 null worms. The graph shows the mating success score (± SD) of each experimental repeat: considering 0% (no mating) or 100% (mating) for each hermaphrodite singled-out from a plate containing males of the strain being tested (a total of N=18 male worms from each strain were analyzed, over 3 independent experiments). 100% of the
ift-20 null males fail to mate with wild-type control hermaphrodites. ****P≤0.0001.