Figure 6. Hybrid cilium position is biased in the direction of beating and dependent on flow.

(A) Left: Cartoon depiction of the strategy for analysis of the position of the hybrid cilium relative to cilia beating direction. Middle: representative 3DSIM image used for data analysis. Scale bar represents 5μm. Right: MATLAB-based analysis strategy to assess rotational polarity in multiciliated cells. The position is calculated relative to coordinates (−0.5<x,y<+0.5) obtained by normalizing the cell length and width to 1. Cell length (y-axis) is assigned as parallel to the direction of ciliary beating measured by rotational polarity of basal bodies-basal feet pairs, cell width (x-axis) is assigned as perpendicular to the direction of ciliary beating. (B) Scatterplot showing the cumulative distribution of hybrid cilia along normalized cell width and cell length in human airway multiciliated cells from three healthy individuals (red) or one PCD patient with immotile cilia (green) caused by loss-of-function mutations in DNAH5 (p.[(Arg478*) and intronic mutation abolishing accepted splice-site for exon 68]), indicating a positional bias toward the direction of ciliary beating (See also two other PCD patients in Figure S8). Each dot represents a hybrid cilium in a cell; Healthy n=694, PCD1 =165, **** p<0.0001, student’s t-test. (C) Scatterplot-bar graphs showing distribution of ratio of hybrid cilium-cell centroid distance to hybrid cilium-cell membrane shortest distance of human airway multiciliated cells from healthy individuals or PCD1 patient. Each dot represents a hybrid cilium in a cell; Healthy n=694, PCD n=277, p<0.0001, student’s t-test. **** p<0.0001. Data are represented as mean ± SD. (D) 3D-SIM volume maximum intensity projection of MTEC labeled with anti-CNTRL (green) antibody, anti-CEP135 (magenta) and anti-ZO1 (blue) subjected to rotational movement to facilitate flow generation in transwells. Arrows indicate CNTRL rings. MTEC ALI, mouse trachea epithelia cells growing at air liquid interface. (E) 3D-SIM volume maximum intensity projection of mouse tracheal tissues labelled with anti-CNTRL (green) antibody and anti-γTub (red). Arrow indicates beating direction. Scale bar represents 10 μm. (F) High-magnification view of boxed area in (E). Note hybrid cilium basal body presenting multiple basal feet harbouring γ-Tubulin labeling. Scale bar represents 1 μm. See also Figure S4.