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...

Centrosome aberrations are commonly observed in human tumors and correlate with tumor aggressiveness and poor prognosis. Extra centrosomes drive mitotic errors that have been implicated in promoting tumorigenesis in mice. However, centrosome aberrations can also disrupt tissue architecture and confer invasive properties that may facilitate the dissemination of metastatic cells. Recent work has shown that centrosome defects facilitate invasion through cell-autonomous and non-cell-autonomous mechanisms, suggesting cancer cells can benefit from centrosome aberrations present in a subset of the tumor cell population. Here, we discuss how centrosome defects promote invasive behaviors that may contribute to initial steps in the metastatic cascade.

Multiciliated cells (MCCs) are terminally differentiated epithelia that assemble multiple motile cilia used to promote fluid flow. To template these cilia, MCCs dramatically expand their centriole content during a process known as centriole amplification. In cycling cells, the master regulator of centriole assembly Polo-like kinase 4 (PLK4) is essential for centriole duplication; however recent work has questioned the role of PLK4 in centriole assembly in MCCs. To address this discrepancy, we created genetically engineered mouse models and demonstrated that both PLK4 protein and kinase activity are critical for centriole amplification in MCCs. Tracheal epithelial cells that fail centriole amplification accumulate large assemblies of centriole proteins and do not undergo apical surface area expansion. These results show that the initial stages of centriole assembly are conserved between cycling cells and MCCs and suggest that centriole amplification and surface area expansion are coordinated events.