Figure 4. Fltp is expressed and necessary during BB docking.

(A) Scheme of multiciliated cell (MCC) maturation. A MCC progenitor projects a primary cilium at the apical surface (stage I). Centrosome amplification is the first sign of differentiation (stage II) followed by apical transport and docking of BBs (stage III). Fully differentiated cells project multiple motile cilia at the apical surface (stage IV). Staging according to Vladar and Stearns (2007). The green nucleus indicates Fltp reporter gene expression. (B–G) LSM of ALI culture of Fltp^ZV and WT mTECs. (B) Onset and level of Fltp reporter expression correlate with the onset of BB amplification and ciliogenesis (stage II–IV) in cultured ALI mTECs as shown in the scheme in (A). (C–C′′′) Confocal sections of a single cell from the sub-apical actin level (C) over the apical actin level (C′ and C″) to the cilia level (C′′′) (ALI day 4). Fltp co-localizes with the MT-plus end binding protein EB-1 (D) (ALI day 4) next to the pericentriolar matrix (E) (ALI day 4) and to cilia in multiciliated Fltp^+/+ cells (C′′′). (F) Fltp^+/+ cell showing all BBs (red dots) projecting cilia (white stripes) at the apical surface (ALI day 4). (G) In many Fltp^ZV/ZV cells the majority of BBs are not docked at the level of tight junctions marked by ZO-1 and do not project cilia (ALI day 4). (H and I) Side view IMARIS surface rendering shows that all BBs are docked at the apical surface in Fltp^+/+ (H) (ALI day 4) in contrast to Fltp^ZV/ZV cells where most BBs stay in the cytoplasm (I) (ALI day 4). BBs are marked by γ-Tubulin (γTub) and pericentrin (Peric), cilia and the tubulin network by tyrosinated-Tubulin (Tyr Tub) and acetylated-Tubulin (aT), the actin network by Phalloidin (Phall), MT plus ends by EB-1, Fltp protein by Fltp116-1, nuclei by DAPI, and nuclei of Fltp reporter expressing cells by GFP. Scale bars; 2 µm (C–C′′′, D, E), 5 µm (F and G).

DOI: http://dx.doi.org/10.7554/eLife.03842.010

Figure 4—figure supplement 1. ALI differentiation efficiency and quantification of BB docking defect.

(A) Graph showing the percentage of cells in stage II–IV for WT and Fltp^ZV/ZV (HOM) ALIs at day zero (before the switch to ALI condition), one, two, and four measured by GFP and pericentrin (HOM) and only pericentrin (WT) staining compared to the total amount of cells in the ALI culture stained by DAPI (representative images were analyzed). For WT analysis we counted n = 1 (1493 cells) for day 0, n = 1 (1345) for day 1, n = 2 (1635) for day 2, n = 2 (1262) for day 4 and for HOM n = 1 (1367 cells) for day 0, n = 1 (1542) for day 1, n = 3 (942) for day 2, n = 4 (1123) for day 4. (B) Shows the percentage of ciliation (measured by acetylated-Tubulin staining) of cells with amplified centrosomes (measured by pericentrin staining) for WT and HOM ALIs at day two and four categorized into stage II–III and stage IV. For WT analysis we counted n = 4 (609 cells) for day 2, n = 4 (231) for day 4 and for HOM n = 10 (611) for day 2, n = 7 (286) for day 4.

DOI: http://dx.doi.org/10.7554/eLife.03842.011