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. 2018 Oct 16;7:e39655. doi: 10.7554/eLife.39655

Figure 1. Role of dynein-2 intermediate chains WDR34 and WDR60 in ciliogenesis.

(A) Cilia stained with the markers Arl13b (green) and acetylated tubulin (AcTub, red) in RPE1 WDR60 and WDR34 KO cell lines. Panels on the right show enlargements of the boxed regions. Scale bars 5 μm. (B) Percentage of ciliated cells (n = 3; 656 WT, 430 WDR34 KO#1, 296 WDR34 KO#2, 397 WDR34 KO CTRL, 343 WDR60 KO and 195 WDR60 KO CTRL cells quantified). (C) Cilium length in WDR60 and WDR34 KO compared with WT cells and CRISPR control cells lines (n = 3; 120 WT, 158 WDR60 KO, 138 WDR60 KO CTRL and 30 WDR34#1 cells quantified). Mann-Whitney test was used, p-value: ****=<0.0001. (D–Hi) Representative 70 nm thick EM sections of (D) WT, (E) WDR34 KO and (F, G– Hi) WDR60 KO cilia. (E) Six serial sections through a WDR34 KO cilium showing no axoneme extension. (G) Two serial sections through a WDR60 KO cilium showing complete cilium. Arrows point to the bulbous ciliary tip and to a membrane protrusion containing membrane vesicles; enlargements are shown to the right (H and Hi). Scale bar length and magnification is indicated on the images.

Figure 1.

Figure 1—figure supplement 1. Generation of a WDR60 and WDR34 KO cell line.

Figure 1—figure supplement 1.

(A and B) Genomic sequence for CRISPR-Cas9 target sites in the two WDR34 KO cell lines (identified as #1 and #2) and (C) WDR60 KO cells. Black lines and scissors depict gRNA binding and cut sites. Nucleotides in green show the CRISPR PAM site. Amino acid translation shown underneath; with the alternative translation in KO cells. Asterisk indicates a premature stop codon. In WDR34 KO cell clone #2 the mutation in the second allele affects the acceptor site of the intron. (D and E) Diagrams of WT type WDR60 and WDR34 and CRISPR mutant proteins. WDR60 is a protein of 1067 aa, in WDR60 KO the stop codon is introduced in exon 2 (76 aa). WDR34 is a protein of 536aa, in WDR34 KO #1 cells a stop codon is introduced in exon 2 (130aa).
Figure 1—figure supplement 2. CRISPR control cells lines show no defect in ciliogenesis.

Figure 1—figure supplement 2.

(A and B) Immunoblotting for WDR34 and WDR60 in WT and KO cells. (A) A 52 kDa band, corresponding to WDR34 is lost in WDR34 KO cells lysate. (B) A WDR60 band of 123 kDa is lost in the WDR60 KO cells lysate. (C) WDR34 KO CTRL and WDR60 KO CTRL cells stained with Arl13b (green) and AcTub (red). (D) WDR60 KO CTRL cells stained with IFT88 (green). (Di) IFT88 intensity quantification in WT and WDR60 KO CTRL cells (n = 3, 98 WT, 102 WDR60 KO CTRL, and 122 WDR60 KO cells quantified). Mann-Whitney test was used, p-value: *=<0.05 as above. Scale bars 5 μm.