Dynamics of centriole amplification in centrosome-depleted brain multiciliated progenitors

Olivier Mercey,Alexia Mahuzier,Amélie-Rose Boudjema,Nathalie Spassky,Philippe Rostaing,Adel Al Jord,Aurélien Fortoul,Alice Meunier,Marion Faucourt

doi:10.1038/s41598-019-49416-2

Abstract

Reproductive and respiratory organs, along with brain ventricles, are lined by multiciliated epithelial cells (MCC) that generate cilia-powered fluid flows. MCC hijack the centrosome duplication pathway to form hundreds of centrioles and nucleate motile cilia. In these cells, the large majority of procentrioles are formed associated with partially characterized organelles called deuterosomes. We recently challenged the paradigm that deuterosomes and procentrioles are formed de novo by providing data, in brain MCC, suggesting that they are nucleated from the pre-existing centrosomal younger centriole. However, the origin of deuterosomes and procentrioles is still under debate. Here, we further question centrosome importance for deuterosome and centriole amplification. First, we provide additional data confirming that centriole amplification occurs sequentially from the centrosomal region, and that the first procentriole-loaded deuterosomes are associated with the daughter centriole or in the centrosomal centriole vicinity. Then, to further test the requirement of the centrosome in deuterosome and centriole formation, we depleted centrosomal centrioles using a Plk4 inhibitor. We reveal unexpected limited consequences in deuterosome/centriole number in absence of centrosomal centrioles. Notably, in absence of the daughter centriole only, deuterosomes are not seen associated with the mother centriole. In absence of both centrosomal centrioles, procentrioles are still amplified sequentially and with no apparent structural defects. They seem to arise from a focal region, characterized by microtubule convergence and pericentriolar material (PCM) assembly. The relevance of deuterosome association with the daughter centriole as well as the role of the PCM in the focal and sequential genesis of centrioles in absence of centrosomal centrioles are discussed.

Highlights

Multiciliated cells (MCC) grow up to several hundred of motile cilia to generate fluid flow necessary for proper respiratory, reproductive, and brain functions[1]
In order to precise early A-phase dynamics, we used our in vitro assay where the differentiation of mouse Centrin 2 GFP-tagged (Cen2GFP) ependymal cells allows the monitoring of centriole amplification dynamics (Fig. 1a,b, Supplementary Movie 1)[8,11]
We quantified that (i) the number of Cen2GFP halos increased over time in all the cells (Fig. 1a,c; grey), confirming, as previously stated[8], that procentrioles are formed sequentially in the brain multiciliated epithelial cells (MCC) progenitor, and (ii) the maturation of procentrioles at the A- to G-phase transition was timely correlated with a stop in the generation of new Cen2GFP structures (Fig. 1a,c,d; orange), confirming that procentriole formation occurs exclusively during A-phase

Summary

Introduction

Multiciliated cells (MCC) grow up to several hundred of motile cilia to generate fluid flow necessary for proper respiratory, reproductive, and brain functions[1]. These cilia are nucleated by centriole-derived basal bodies docked at the plasma membrane. Because two centrosomal centrioles seemed insufficient to scaffold the formation of tens of centrioles, massive centriole production through deuterosome structures was proposed to arise independently from the centrosome in MCC6 Challenging this postulate, an electron microscopy study showing association of deuterosomes with centrosomal centrioles in chick trachea proposed that the “procentriole clusters may form initially in close association with the diplosomal centrioles”[7]. Using new tools available to study centriole amplification in MCC –a home-made Deup[1] antibody and the Plk[4] inhibitor centrinone- we further investigate the relationship between the centrosome organelle and the dynamics of centriole amplification by (i) characterizing the centrosome behavior during centriole amplification and (ii) assessing the dynamics of amplification in cells depleted from one or both centrosomal centrioles

Methods

Results

Conclusion