Journal: Nature Communications

Article Title: Directional ciliary beats across epithelia require Ccdc57-mediated coupling between axonemal orientation and basal body polarity

doi: 10.1038/s41467-024-54766-1

Figure Lengend Snippet: Quantification results in ( d , e , and g ) are presented as mean ± SD plus sample dots. P -values were from unpaired two-tailed student’s t -test. *** P < 0.001. a Illustration for the beat pattern of an ependymal cilium (side views). b Experimental setup. Cilia motilities were imaged with a spinning disk confocal microscope for SiR-tubulin-labeled cilia alone ( c ) or together with fluorescent beads that served as tracers of liquid flows ( h , i ). Note that cilia recorded in this way are mostly top views. c Beat dire c tions of multicilia (arrows) in individual E1 cells of representative microscopic fields. Images were acquired at 10 ms intervals on ependymal tissues from P35 littermates. The first three consecutive frames, cropped from Supplementary Movies and , were pseudo-colored and merged to show ciliary motilities. Representative fields from other littermates are presented in Supplementary Fig. 3d. d Percentages of multicilia displaying directional beats, quantified from three mice of each genotype described in ( c ). 200 multi c iliary bundles were scored per mouse. e Quantification of mean vector length from 9 microscopic fields from three mice of each genotype described in ( c ). P = 4.7 × 10 −6 . The mean vector length is 1 when all multiciliary bundles in a field beat in the same direction or 0 when randomly. f Image sequences showing directional ciliary beat in individual Ccdc57 −/− E1 cells, cropped from Supplementary Movies and to cover three beat cycles. Effective and recovery strokes are indicated by colored arrows. Refer to Supplementary Fig. 3c, d for additional examples. g Ccdc57 deficiency did not alter multicilia beat frequencies. Quantification from three mice per genotype (50 muliticilary bundles per mouse) was pooled. h , i Ccdc57 deficiency impaired multicilia-driven liquid flows. The first three consecutive frames cropped from Supplementary Movies and were merged to show ciliary distributions and motilities. The first corresponding frames of beads were superimposed with trajectories of traceable, rapidly moving beads in the first 858 ms to show flow directions ( h ). In ( i ), all frames of beads were projected together to show flow patterns. Trajectories of traceable, rapidly moving beads in the first 300 ms (P47) or 400 ms (P96) were superimposed to show flow directions. Source data are provided as a Source Data file.

Article Snippet: The skeletons were scanned with a high-resolution 3D X-ray microscope based on micro-computed tomography (micro-CT) technology (Bruker, SkyScan 1272) at a resolution of 9 μm.

Techniques: Two Tailed Test, Microscopy, Labeling, Plasmid Preparation