A role for Cep70 in centriole amplification in multiciliated cells

International audienceCentriole amplification in multiciliated cells occurs in a pseudo-cell cycle regulated process that typically utilizes a poorly characterized molecularly dense structure called the deuterosome. We identified the centrosomal protein Cep70 as a novel deuterosome-associated protein that forms a complex with other deuterosome proteins, CCDC78 and Deup1. Cep70 dynamically associates with deuterosomes during centriole amplification in the ciliated epithelia of Xenopus embryos. Cep70 is not found in nascent deuterosomes prior to amplification. However, it becomes localized at deuterosomes at the onset of centriole biogenesis and remains there after the completion of centriole amplification. Deuterosome localization requires a conserved C-terminal "Cep70" motif. Depletion of Cep70 using morpholino oligos or CRISPR/Cas9 editing in F0 embryos leads to a severe decrease in centriole formation in both endogenous MCCs, as well as ectopically induced MCCs. Consistent with a decrease in centrioles, endogenous MCCs have defects in the process of radial intercalation. We propose that Cep70 represents a novel regulator of centriole biogenesis in MCCs

Massive centriole production can occur in the absence of deuterosomes in multiciliated cells

International audienceMulticiliated cells (MCCs) amplify large numbers of centrioles that convert into basal bodies, which are required for producing multiple motile cilia. Most centrioles amplified by MCCs grow on the surface of organelles called deuterosomes, whereas a smaller number grow through the centriolar pathway in association with the two parent centrioles. Here, we show that MCCs lacking deuterosomes amplify the correct number of centrioles with normal step-wise kinetics. This is achieved through a massive production of centrioles on the surface and in the vicinity of parent centrioles. Therefore, deuterosomes may have evolved to relieve, rather than supplement, the centriolar pathway during multiciliogenesis. Remarkably, MCCs lacking parent centrioles and deuterosomes also amplify the appropriate number of centrioles inside a cloud of pericentriolar and fibrogranular material. These data show that the centriole number is set independently of their nucleation platforms and suggest that massive centriole production in MCCs is a robust process that can self-organize

Lack of GAS2L2 Causes PCD by Impairing Cilia Orientation and Mucociliary Clearance

International audiencePrimary ciliary dyskinesia (PCD) is a genetic disorder in which impaired ciliary function leads to chronic airway disease. Exome sequencing of a PCD subject identified an apparent homozygous frameshift variant, c.887_890delTAAG (p.Val296Glyfs∗13), in exon 5; this frameshift introduces a stop codon in amino acid 308 of the growth arrest-specific protein 2-like 2 (GAS2L2). Further genetic screening of unrelated PCD subjects identified a second proband with a compound heterozygous variant carrying the identical frameshift variant and a large deletion (c.867_∗343+1207del; p.?) starting in exon 5. Both individuals had clinical features of PCD but normal ciliary axoneme structure. In this research, using human nasal cells, mouse models, and X.laevis embryos, we show that GAS2L2 is abundant at the apical surface of ciliated cells, where it localizes with basal bodies, basal feet, rootlets, and actin filaments. Cultured GAS2L2-deficient nasal epithelial cells from one of the affected individuals showed defects in ciliary orientation and had an asynchronous and hyperkinetic (GAS2L2-deficient = 19.8 Hz versus control = 15.8 Hz) ciliary-beat pattern. These results were recapitulated in Gas2l2-/- mouse tracheal epithelial cell (mTEC) cultures and in X. laevis embryos treated with Gas2l2 morpholinos. In mice, the absence of Gas2l2 caused neonatal death, and the conditional deletion of Gas2l2 impaired mucociliary clearance (MCC) and led to mucus accumulation. These results show that a pathogenic variant in GAS2L2 causes a genetic defect in ciliary orientation and impairs MCC and results in PC