In situ cryo-electron tomography reveals the progressive biogenesis of basal bodies and cilia in mouse ependymal cells.

Cilia, essential organelles for cell motility and signaling, comprise an axoneme extended from the basal body (BB). The assembly process of BBs and axonemes during ciliogenesis, however, remains largely unknown due to the lack of structural information. Here, we leverage in-situ cryo-electron tomography to capture within mouse ependymal cells the dynamic processes of BB biogenesis and multiciliogenesis at various stages. This approach enables 3D visualization of the complete motile machinery, revealing the continuous microtubule-based scaffold from BBs to axonemes at sub-nanometer resolution with unprecedented structural details. Furthermore, we elucidate along BBs and cilia heterogeneous landscapes of microtubule-binding proteins underlying the establishment of structural periodicity and diverse subregions. Notably, the chronological binding patterns of microtubule-inner proteins (e.g., CEP41) correlate with the progressive assembly of ciliary beating machinery. We also resolve a substructure that borders the BB and the axoneme. Our findings provide key insights into intricate orchestrations during ciliogenesis.