Inherited retinal degeneration due to loss of photoreceptor cells is a leading cause of human blindness. These cells possess a photosensitive outer segment linked to the cell body through the connecting cilium (CC). While structural defects of the CC have been associated with retinal degeneration, its nanoscale molecular composition, assembly, and function are barely known. Here, using expansion microscopy and electron microscopy, we reveal the molecular architecture of the CC and demonstrate that microtubules are linked together by a CC inner scaffold containing POC5, CENTRIN, and FAM161A. Dissecting CC inner scaffold assembly during photoreceptor development in mouse revealed that it acts as a structural zipper, progressively bridging microtubule doublets and straightening the CC. Furthermore, we show that Fam161a disruption in mouse leads to specific CC inner scaffold loss and triggers microtubule doublet spreading, prior to outer segment collapse and photoreceptor degeneration, suggesting a molecular mechanism for a subtype of retinitis pigmentosa.

Vertebrate multiciliated cells (MCCs) contribute to fluid propulsion in several biological processes. We previously showed that microRNAs of the miR-34/449 family trigger MCC differentiation by repressing cell cycle genes and the Notch pathway. Here, using human and Xenopus MCCs, we show that beyond this initial step, miR-34/449 later promote the assembly of an apical actin network, required for proper basal bodies anchoring. Identification of miR-34/449 targets related to small GTPase pathways led us to characterize R-Ras as a key regulator of this process. Protection of RRAS messenger RNA against miR-34/449 binding impairs actin cap formation and multiciliogenesis, despite a still active RhoA. We propose that miR-34/449 also promote relocalization of the actin binding protein Filamin-A, a known RRAS interactor, near basal bodies in MCCs. Our study illustrates the intricate role played by miR-34/449 in coordinating several steps of a complex differentiation programme by regulating distinct signalling pathways.

ulticiliated cells (MCCs) contain tens of motile cilia that beat to drive fluid flow across epithelial surfaces. Multiciliated cells are present in the respiratory tract, brain ventricles and reproductive systems. Defects in motile-cilia formation or beating lead to the development of hydrocephaly, lethal respiratory symptoms and fertility defects 1-4. A centriole, or basal body, serves as a template for the cilium axoneme. Centriole duplication is tightly controlled in cycling cells so that a single new procentriole forms adjacent to each of the two parent centrioles 5. However, MCC progenitors with two parent centrioles produce tens to hundreds of additional new centrioles to nucleate multiple motile cilia 1. Steric constraints imposed by the 'centriolar' pathway seem to restrict the number of procentrioles that can be nucleated by the parent centrioles. Centriole amplification is therefore thought to rely on the assembly of dozens of MCCspecific organelles called deuterosomes, which each nucleate tens of procentrioles 6-14. Deuterosomes are assembled during centriole amplification and support the growth of approximately 90% of the procentrioles formed in mammalian MCCs 12,14. Deuterosomes have been proposed to be nucleated from the younger parent centriole 12 but can form spontaneously in a cloud of pericentriolar material (PCM) in MCCs depleted of the parent centrioles 15-17. Many of the proteins required for centriole formation in MCCs are common to centriole duplication 11-15,18-23. However, DEUP1 (CCDC67, alternate gene name) has been identified as a deuterosome-specific protein that arose from a gene-duplication event of the centriolar gene Cep63. Recent data suggest that Deup1 evolved to enable the formation of deuterosomes and the generation of large numbers of centrioles 14. In this manuscript, we interrogate the function of the deuterosome in MCCs from mouse and from Xenopus laevis. Surprisingly, our findings reveal that deuterosomes are dispensable for centriole amplification and multiciliogenesis both in vitro and in vivo. Moreover, we show that neither deuterosomes nor parent centrioles are required for MCCs to amplify the correct number of centrioles. These findings raise new questions about the evolutionary role of deuterosome during multiciliogenesis and the mechanisms regulating centriole number in MCCs. Results Generation of a Deup1-knockout mouse. To examine the role of the deuterosome in multiciliogenesis we created a Deup1-knockout mouse by replacing a region from within exon 2 to within exon 7 of the Deup1 gene with a LacZ reporter (Extended Data Fig. 1a). Reverse Transcription-quantitative PCR on brain and testes samples showed that the messenger RNA levels of Deup1 were reduced by at least tenfold in Deup1-knockout compared with control mice (Extended Data Fig. 1b,c). To examine the process of multiciliogenesis in Deup1 −/− cells, we utilized in vitro cultures of mouse tracheal epithelial cells (mTECs) or ependymal cells 24,25. Consistent with the absence of Deup1 mRNA, DEUP1 foci were a...

Multiciliated cells (MCCs) harbor dozens to hundreds of motile cilia, which generate hydrodynamic forces important in animal physiology. In vertebrates, MCC differentiation involves massive centriole production by poorly characterized structures called deuterosomes. Here, single-cell RNA sequencing reveals that human deuterosome stage MCCs are characterized by the expression of many cell cycle-related genes. We further investigated the uncharacterized vertebrate-specific cell division cycle 20B (CDC20B) gene, which hosts microRNA-449abc. We show that CDC20B protein associates to deuterosomes and is required for centriole release and subsequent cilia production in mouse and Xenopus MCCs. CDC20B interacts with PLK1, a kinase known to coordinate centriole disengagement with the protease Separase in mitotic cells. Strikingly, over-expression of Separase rescues centriole disengagement and cilia production in CDC20B-deficient MCCs. This work reveals the shaping of deuterosome-mediated centriole production in vertebrate MCCs, by adaptation of canonical and recently evolved cell cycle-related molecules.