Figure 7.

TTC12 Is Required for the Assembly of Single-Headed IDAs

(A) TTC12 was invalidated in AEC cultures with TTC12 CRISPR-Cas9 lentiviruses and immunostained with an anti-acetylated α-tubulin antibody (red), a marker of ciliary axonemes, and anti-DNAH6, anti-DNAH12, or anti-DNALI1 antibodies as markers of single-headed IDAs (green). IF analysis in clusters of ciliated cells shows the partial invalidation of TTC12 in culture with a loss of DNAH6, DNAH12, and DNALI1 in cells lacking TTC12. Scale bars represent 25 μm. DNAH6-, DNAH12-, and DNALI1-labeled areas were measured with ImageJ software and normalized against tubulin-labeled area. The dynein/tubulin ratios obtained in TTC12-CRISPR-transduced cells were then normalized to ratios obtained in EV-transduced cells. Values of those final ratios are indicated on the right of the histograms and showed the decrease of dynein-chain-labeling signal compared to tubulin. Data are represented as the mean ± SEM. Statistical analyses were performed with GrahPad Prism v5 software and two-tailed unpaired t tests. ^∗∗∗p < 0.001 (n = 3).

(B) IF images of isolated ciliated cells lacking TTC12 show the complete loss of DNAH12 along the cilia and its accumulation in the subapical region of the cytoplasm, whereas DNAH6 and DNALI1 expression appears to be strongly decreased but still present at the base of cilia. Nuclear staining was performed with DAPI (blue). The scale bar represents 25 μm.

(C) Electromicrographs of cross-sections of cilia axonemes from a non-infected AEC culture and a TTC12-CRISPR-infected cell culture show an abnormal arrangement lacking IDAs in the TTC12-invalidated culture. Proportions of cilia harboring more than four IDAs, between two and four IDAs, and up to one IDA,in electromicrograph cross-sections, were reported in a histogram comparing control cells to TTC12-invalidated cells. Scale bars represent 5 μm.

Abbreviations are as follows: ODA, outer dynein arm; IDA, inner dynein arm; and EV, empty vector.