Figure 4.

Human CDKL5 Localizes to Cilium and Affects Ciliogenesis When Overexpressed in RPE-1 Cells and Mutations Modeled in C. elegans CDKL-1 Disrupt Localization and/or Cilium Length Regulation

(A) Immunofluorescence analysis of serum-starved hTERT RPE-1 cells expressing GFP-CDKL5 stained with antibodies against GFP, ARL13B (cilium marker), and PCNT (centrosome marker). The top and bottom panels are representative images showing different levels of GFP-CDKL5 localization to the BB and cilium tip. Scale bar, 5 μm.

(B) Serum-starved hTERT RPE-1 GFP-CDKL5 cells were stained with antibodies against GFP, IFT88, and polyglutamylated tubulin (centriole/BB and axoneme marker). Scale bar, 5 μm.

(C) Bar graph shows mean percentage of ciliated cells (n > 300 cells per sample, 3 independent experiments) in the serum-starved populations (72 hr) of hTERT RPE-1 and hTERT RPE-1 GFP-CDKL5 cells transfected with the indicated siRNAs. Error bars indicate SD. ^∗∗p < 0.01 (Student’s two-tailed t test).

(D) WT CDKL-1A and the P169L variants localize to the TZ, whereas the G11R mutant localizes along the length of cilia, and the L210P mutant accumulates in dendrites (den) and is weakly present at the periciliary membrane compartment (PCMC). MKS-2 is a TZ marker. Scale bar, 4 μm.

(E) ADL cilia lengths (L4 larvae) of WT and strains expressing CDKL-1 variants. Expressing WT CDKL-1A leads to short cilia. Similar levels of length reduction are not seen upon expression of the G11R and L210P variants, or lines 1 and 2 of the P169L variant (line 3 is not significant), suggesting functional defects with these variants. Dot is one cilium. Significance (p value) was calculated by Dunn Kruskal-Wallis multiple comparison (Holm-Sidak adjustment). ^∗p < 0.01 and ^∗∗p < 0.001; ns, not significant.