Fig. 3: Ependymal cilia display local protein synthesis.

Relative intensities of ciliary Puro were from three independent experiments. The bars and errors represent mean ± s.d. Two-tailed student’s t-test. a Diagram illustrating metabolic labeling of nascent peptides with Puromycin (Puro). Puromycylated peptides were visualized with an anti-Puromycin antibody. The E-, P-, and A-sites in the ribosome are depicted. b, c Protein translation-dependent Puro IF signals were readily detected in multicilia. Day-10 mEPCs were pulse-labeled with Puro for 5 min with or without Cycloheximide (CHX) and then processed for confocal imaging (b). Rsph4a, a radial spoke subunit, served as multiciliary marker⁷¹. The framed region was magnified to show details. In c, 150 multiciliated mEPCs were quantified in each condition. Ciliary intensity of Puro was normalized to that of Rsph4a. d Diagram illustrating the isolation of multicilia-containing apical cortexes. e Multicilia of the isolated cortexes retained translation machinery components and basal bodies. Ac-tub and Odf2 served as ciliary and basal body markers, respectively. f Protein translation-dependent Puro IF signals were detected in isolated multicilia. Pulse labeling was performed as in b. g Diagram illustrating the isolation of ciliary shafts. Multiciliated mEPCs were cultured to day 10 in flasks. After the culture medium was replaced with Ca²⁺-containing deciliation buffer²³, the flasks were shaken horizontally to release individual ciliary shafts. The ciliary shafts in the supernatant were then spun onto poly-L-lysine-coated coverslips for further analyses. h, i Isolated ciliary shafts displayed local protein translation. Isolated ciliary shafts attached to poly-L-lysine-coated coverslips were pulse-labeled with Puro for 5, 15, and 30 min with or without CHX. Mock-treated samples served as negative control. After fixation, the samples were immunostained for Rsph4a and subjected to confocal imaging (h). In i, 300 cilia were quantified in each condition. For each ciliary shaft, the total ciliary IF intensity of Puro was normalized to that of Rsph4a.