Cilia stability and structure are impaired in <i>Ttbk2</i>^{<i>sca11/gt</i>} cilia.

(A-B) Polyglutamylated tubulin localization in cilia of MEFs derived from embryos of the indicated genotypes. Immunostaining for GT335, which recognizes polyglutamylated tubulin (magenta) as well as PCNT (red) to label centrosomes and Acetylated α-Tubulin (green) to label cilia is shown in (A). A quantification of the pixel intensity of GT335 within cilia is shown in (B). Polyglutamylated tubulin levels are reduced in Ttbk2sca11/gt relative to the other genotypes of the allelic series (pTtbk2gt/+; pTtbk2gt/gt; p = 0.0327 vs Ttbk2null/gt). Scale bar = 5μm. (C-D) Wild type, Ttbk2null/gt, and Ttbk2sca11/gt MEFs were treated with 10μM Nocodazole for 10–30 minutes and percentage of ciliated cells was determined. While in WT and Ttbk2null/gt cells the percentage of ciliated cells reduced only slightly within 30 minutes, cilia were rapidly lost in Ttbk2sca11/gt MEFs upon treatment with nocodazole (p = 0.0112) (C). Cilia length was determined throughout nocodazole treatment for Wild type, Ttbk2null/gt, and Ttbk2sca11/gt. As expected, cilia shortened over time, though not significantly between genotypes over time. n = 50 cilia pooled from 3 biological replicates. (E-H) Representative Transmission Electron Microscopy (TEM) images of cilia in the embryonic neural tube from e10.5 embryos of the indicated genotypes. Additionally, vesicles can be seen in Ttbk2sca11/gt axonemes (arrow heads, H) which were not present in other genotypes. Scale bar = 0.5μm.